Compositions and methods for restoring the stratum corneum and treating dermatological diseases

ABSTRACT

The present invention provides methods of treating humans and animals with terpenes and terpenoids in order to restore the stratum granulosum and stratum corneum and treat a dermatological disease, such as, for instance atopic dermatitis. More specifically it provides terpenes and terpenoids such as monoterpenoids, sesquiterpenes and norisoprenoids as ingredients that work to restore and thicken the stratum granulosum and stratum corneum and treat a dermatological disease. Terpenes and terpenoid compounds support the cell proliferation, cell differentiation, growth of skin, skin tissue and keratinocytes by upregulating genes for loricrin filaggrin, caspase 14, hornerin and other late cornified envelope components and vascular endothelial growth factor (VEGF) and downregulation of matrix metalloproteinases. Both terpenes and terpenoids may be applied topically or orally at oral or topical concentrations from about 0.6 mg to about 60 mg per kilogram of body weight per day effective to stimulate growth of the skin layer and prevent damage from ultraviolet radiation (UVR).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2012/040999, filed Jun. 6, 2012, which claims the benefit ofpriority to U.S. Provisional Application No. 61/520,254 filed Jun. 7,2011, the disclosures of which are herein incorporated by reference intheir entireties. Applicants claim the benefit of 35 U.S.C. § 120 as tothe PCT application and the United States provisional application.

FIELD OF THE INVENTION

The present invention relates to methods of treating humans and animalswith terpenes and terpenoids in order to restore the stratum corneum,stratum lucidium and stratum granulosum and treat such water barrierdependent dermatological diseases as atopic dermatitis, eczema, etc.More specifically it relates to the use of terpenes and terpenoids, suchas isoprenoids, monoterpenes, monoterpenoids, sesquiterpenes andnorisoprenoids as ingredients that restore the stratum corneum andrelated structures and are effective to treat such water barrierdependent dermatological diseases as atopic dermatitis, eczema, etc.

BACKGROUND OF THE INVENTION

Terpenes and terpenoids represent a large complex class of compoundsbuilt upon the isoprene unit, C₅H₈. Some terpenes and terpenoids havebeen used in the food, flavor and fragrance industries. Typically theyare of plant or microbial origin and are often bound to tannins orglycosides.

Terpenes, which are important contributors to many fruit and floralscents, are synthesized from geranyl diphosphate (GDP), an intermediatein carotenoid biosynthesis. Monoterpene perillyl alcohol has beenreported to inhibit photocarcinogenesis in a non-melanoma model of mouseskin carcinogenesis and UVB-induced skin carcinogenesis. Perillylalcohol caused a reduction in UVB-induced non-melanoma tumors(Barthelman et al., Cancer Res. 1998; 58:711-716). Topical pretreatmentbut not post-treatment of the melanoma cells with perillyl alcoholmarkedly reduced levels of UV-induced reactive oxygen species. Thesestudies suggest that perillyl alcohol inhibits the Ras signaling pathwayand thus can be an effective target for chemoprevention of melanoma(Lluria-Prevatt et al., Cancer Epidemiology, Biomarkers & Prevention,2002; 11(6):573-579).

Numerous studies have shown major changes in gene expression patternsassociated with terpene exposure (Nakamura et al., FEBS Letters, 2004;572(1-3): 245-250; Einbond et al., Anticancer Research, 2007; 27(2):697-712; Rahman et al., Cancer Research, 2006; 66(9): 4952-4960). Veryfrequently gene expression changes involve clusters of genes directly orindirectly involved in lowering rates of cell proliferation, increasingantioxidative enzymes and increasing apoptosis. Some terpenes can actdirectly on epithelial cells in vitro and produce many of the sameresponses seen in vivo (Kim et al., Molecular Cancer Therapeutics, 2002;1(3): 177-184). Some triterpenes exhibited cancer preventative capacityin breast, uterus, and lung cancer, respectively (Niwa et al., OncologyReports, 2007; 17(3): 617-622; Liby et al., Cancer Research, 2007;67(6): 2414-2419). Extract of a plant Rabdosia rubescens was reported toprevent cancer due to its content of diterpenoids (Ho et al., U.S. Pat.No. 7,351,739, 2008). Another plant extract from Acacia victoriaecontaining saponins suggested that it is the triterpenes which exhibitedexhibit potent anti-tumor effects against a variety of tumor cells(Arntzen et al., US Patent Publication US2006/0073222 A1).

Kunsch et al., U.S. Pat. No. 7,247,714 suggests that certain compoundscan activate cytoprotective response elements (CPREs) that have beenidentified to induce, coordinate and activate certain genes that protectcells from the potentially damaging effects of oxidative stress. CPREshave the DNA consensus sequence 5′-RTGACWNAGCANW-3′, wherein R=A or G,W=A or T, and N=A, G, C or T. The cellular functions of CPRE regulatedgenes are diverse, however in general, these genes act in variouscapacities to regulate and maintain redox homeostasis in the cell. TheCPRE is unique, and different from the antioxidant response element(ARE) and the maf-recognition element (MARE).

d-Limonene is a cyclic monoterpene that can serve as a precursor to ahost of other oxygenated monocyclic monoterpenes such as carveol,carvone, menthol, perillyl alcohol and perillaldehyde. It ischaracterized as a non-nutritive dietary component. d-Limonene is foundin the essential oils of citrus fruits, cherry, mint and herbs. It iswidely used in foods, flavors, fragrances, house hold products and skincleansing formulation. d-Limonene is metabolized to oxygenatedmetabolites in rats and in humans. In rats, the two major serummetabolites of d-limonene are perillic acid and dihydroperillic acid.d-Limonene and its metabolites are detectable in serum, liver, lung andmany other tissues with higher concentrations detected in adipose tissueand mammary gland than in less fatty tissues (Crowell et al., CancerChemother. Pharmacol. 1992; 31: 205-212). Humans produce these two serummetabolites as well as limonene-1,2-diol (Crowell et al., CancerChemother. Pharmacol. 1994; 35: 31-37). Metabolism of perillyl alcoholand d-limonene are similar.

Nootkatone is a sesquiterpene, present in grapefruit, tangerine, orangejuices and oils. It does not appear to be toxic, at least at theconcentrations found in normal strength grapefruit juice (approximately0.0035%, or 35 ppm).

Norisoprenoid beta-damascenone is a widely used fragrance and flavormaterial present in a wide variety vegetables and teas. It is found alsoin grapes and wine either glycosylated or as an aglycon. Between 1 and10 metric tons are used annually in the flavor and fragrance industry.No phototoxicity or photoallergenity has been reported in humans aftertopical application of beta-damascenone and UV-radiation. No skinirritation was observed in mice, guinea pigs and humans up to 5%. Higherconcentration than 5% has not been tested. The LD₅₀ was reported to begreater than 2.0 g/kg (Lapczynski et al., Food and Chemical Toxicology,2007; 45(1): S172-S178).

Natural and synthetic vitamin A derivatives (retinoids) have been usedextensively in the treatment of a variety of skin disorders and havebeen used as skin repair or renewal agents. Vitamin A is a diterpenoid.Compounds such as retinol occur naturally in the human body and areessential for normal epithelial cell differentiation. Retinoic acid, forexample, has been used to treat a variety of skin conditions such as,for instance, acne, wrinkles, psoriasis, age spots and discoloration(Rollman, et al., Arch Dermatol Res., 1985a; 278(1):17-24 and Rollman,et al., Br J. Dermatol., 1985b; 113(4): 405-413; Lowe, et al., PharmacolSkin 1989; 3:240-248). Although retinoids efficiently arrest the cellcycle of many types of epithelial tumors during the G₁ stage, no singlecommon mechanism of action has been identified. Retinal (preformedvitamin A) and/or certain carotenoids (provitamin A) are converted toretinol in the body, as needed. Retinol and the other retinoids areintegrally involved in cell growth and differentiation, which may affectcarcinogenesis, but retinoids often prove to be toxic at efficaciouslevels.

Vitamin C (L-ascorbic acid) has been described for use in topicaladministration to reinforce the cohesion of the dermo-epidermal junction(Bernerd, F. U.S. patent application Ser. No. 10/358,888, 2003).Pharmacologically, ascorbate ion is an antioxidant which is required ascofactor in collagen synthesis. Some studies suggested that ingestion ofvitamin C rejuvenates skin. The same was observed with omega-6-linolenicacid.

The skin protects against external injuries such as wounding, UVradiation and microorganisms. It also is a water impermeable barrieragainst dehydration. The major barrier resides in the upper layer of theepidermis. This epidermis is continuously renewed. The basalkeratinocytes are formed first in the proliferating layer stratumbasale. These basal keratinocytes undergo terminal differentiation andbecome corneocytes in the outer layer of epidermis. This process iscalled cornification. Corneocytes reinforce their cytoskeletal keratinfilament network in the stratum spinosum. Later these keratinocytesbecome more flat and express proteins such as profilaggrin and loricrinwhich aggregate and create keratohyalin granules of the stratumgranulosum. At the final stage of differentiation the keratinocytesloose their nucleus and organelles and become dead. Protein profilaggrinis proteolytically modified in to filaggrin by Caspase 14. Fillagrinundergoes further proteolytic degradation with exo- and endo-proteasesinto hygroscopic amino acids which are responsible for the hydration ofthe skin. (Denecker et al., 2008) Another major protein of the cornifiedcell envelope of the terminally differentiated keratinocytes is loricrinwhich is eventually crosslinked to the keratin of the corneum. Loricrincontributes about 70% to the protein mass of the envelope cells, but isa minor percentage of the protein mass of the corneum (Candi et al.,Nature Reviews Molecular Cell Biology 2005; 6: 328-340).

The skin renewal process relies on three events: (1) proliferation ofnew cells in the stratum basale, (2) cytoskeletal reinforcement instratum spinosum and (3) differentiation of keratinocytes in stratumgranulosum and stratum corneum. In the transitional layer between thestratum granulosum and the stratum corneum, lipids are extruded to forma water repelling envelope around the cornified envelope, therebyassuring an adequate permeability barrier function of the mammalianepidermis. Improper formation of these envelopes results in an impairedepidermal barrier that cannot protect against dehydration, UVB, andinfection. Expression of late differentiation markers such as loricrinand filaggrin takes place at the end of the stratum granulosum.Expression of caspase 14 is crucial for proteolytic modification anddegradation of profilaggrin. Caspase 14 is first expressed as procaspase14 and it is activated at the interface between the granular andcornified layer. Once the profilaggrin is modified into fillaggrin,other proteases further process filaggrin into hygroscopic amino acids.Caspase 14 is also essential in protection against UVB-induced damage.How caspase 14 establishes UVB filtering capacity of the corneum is notcompletely understood.

The stratum corneum is the outermost layer of the epidermis, composed oflarge, flat, polyhedral, plate-like envelopes filled with keratin, whichis made up of dead cells that have migrated up from the stratumgranulosum. The stratum corneum is composed mainly of dead cells thatlack nuclei. As these dead cells slough off on the surface in the thinair-filled stratum disjunctum, they are continuously replaced by newcells from the stratum germinativum (basale). In the human forearm, forexample, about 1300 cells/cm²/hr are shed. This outer layer that issloughed off is also known as the stratum dysjunctum. Cells of thestratum corneum contain keratin, a protein that helps keep the skinhydrated by preventing water evaporation. These cells can also absorbwater, further aiding in hydration, and explaining why humans and otheranimals experience wrinkling of the skin on the fingers and toes(“pruning”) when immersed in water for prolonged periods. In addition,this layer is responsible for the “spring back” or stretchy propertiesof skin. A weak glutenous protein bond pulls the skin back to itsnatural shape.

The thickness of the stratum corneum varies according to the amount ofprotection and/or grip required by a region of the body. For example,the hands are typically used to grasp objects, requiring the palms to becovered with a thick stratum corneum. In a similar manner, the sole ofthe foot is prone to injury, and so it is protected with a thick stratumcorneum layer. In general, the stratum corneum contains 15 to 20 layersof dead cells. The stratum corneum has a thickness between 10 and 40 μm.

The major proteins of the cornified cell envelope of the terminallydifferentiated keratinocytes are filaggrin, loricrin, keratin and othercornified envelope gene products, all of which are eventuallycrosslinked with the keratin of the corneum. Filaggrin, loricrin andother cornifying gene products contribute about 70% to the protein massof the envelope, but the percentages may vary in the corneum (Candi etal., Nature Reviews Molecular Cell Biology 2005; 6: 328-340).

A VEGF, or vascular endothelial growth factor, that represents in theskin a major angiogenesis factor, is downregulated after UV-lightexposure. Matrix metalloproteinases (MMPs) are enzymes that degrade theextracellular matrix in the context of physiological remodeling of theskin, but age and exposure to UV radiation have the effect of increasingthe activity of these MMPs, in particular that of MMP1, MMP3 and MMP9.

UVR causes skin inflammation and redness (erythema) associated withsunburn. Sunscreen products typically applied in the form of a creamconsist of active ingredients that adsorb UV-rays directly. Suitablesunscreens can have UVA absorbing properties, UVB absorbing propertiesor a mixture thereof. The exact amount of the sunscreen activeingredient may vary depending upon the desired Sun Protection Factor,i.e. the “SPF” of the composition as well as the desired level of UVRprotection. SPF is a commonly used measure of photoprotection of asunscreen against erythema. The SPF is defined as a ratio of theultraviolet energy required to produce minimal erythema on protectedskin to that required to produce the same minimal erythema onunprotected skin in the same individual (See, Federal Register, 43(166):38206-38269, Aug. 25, 1978). Suitable sunscreens include, but are notlimited to, those found in the CTFA International Cosmetic IngredientDictionary and Handbook, 7^(th) edition, volume 2 pp. 1672, edited byWenninger and McEwen (The Cosmetic, Toiletry, and Fragrance Association,Inc., Washington, D.C., 1997).

Suitable UVA absorbing sunscreen actives include, for instance,dibenzoylmethane derivatives, anthranilate derivatives such asmethylanthranilate and homomethyl, 1-N-acetylanthranilate, and mixturesthereof. Examples of dibenzoylmethane sunscreen actives are described inU.S. Pat. No. 4,387,089 issued to Depolo; and in Sunscreens:Development, Evaluation, and Regulatory Aspects edited by N. J. Lowe andN. A. Shaath, Marcel Dekker, Inc (1990). These are all UVR absorbingtype of sunscreen providing broad-spectrum UVA protection eitherindependently, or in combination with, other UV protective actives thatmay be present in the composition.

Terpenes display some very interesting properties related to cancerprevention (Morita et al., Immunological Reviews 2007; 215: 59-76; Libyet al., Cancer Research, 2007; 67(6): 2414-2419; Niwa et al., OncologyReports, 2007; 17(3): 617-622; Sengottuvelan et al., Carcinogenesis,2006; 27(5): 1038-1046). Their relative safety makes them suitable foruse in flavors and fragrances. Sometimes stereo-isomers of the sameterpene have entirely different sensory profiles. Carvone is an example:4R-(−)-carvone exhibits sweet spearmint flavor, whereas 4S-(+)-carvonetastes like caraway. Terpenes also tend to exhibit relatively highhydrophobicity and some are perceived as bitter to humans.Hydrophobicity allows terpenes to interact directly with cell membranesto trigger responses, such as greater level of anti-oxidation andapoptosis. Terpenes such as limonene, farnesol, geraniol and nerolidolhave been suggested to enhance transdermal and transmucosal drugdelivery (e.g. Aqil et al., Drug Discovery Today 2007; 12(23-24):1061-1067). The penetration of terpenes is linked to their size,hydrophobicity, chirality, saturation and boiling point.

It seems that the receptor sites for bitter substances on the tastecells tends to be hydrophobic (Katsuragi et al., Biochim Biophys Acta1996; 1289: 322-328) and may provide a mechanism whereby terpenes caninteract directly with cell membranes to trigger responses, such asgreater levels of anti-oxidation and apoptosis. Two additional importantcharacteristics of terpene flavors are intensity and thresholdconcentration which are determined by the specificity of receptors (Ruizet al., Chemical Senses 2006; 31(9):813-820). Terpenes are useful asskin penetration enhancers and agents involved in the prevention andtherapy of inflammatory diseases. Some terpenes are better at preventingcancer than others and some can extend lifespan in lower organisms inaddition to preventing cancer. Activity generally tends to increase withthe order of polyterpenes and also hydroxylation (Crowell et al.,Carcinogenesis 1992; 13(7): 1261-1264). Because of variable tastes andtaste intensities and documented utility as dietary inhibitors ofcarcinogenesis, the huge variety of available terpenes provides afertile field for establishing how they might be useful in sunburnprotection and cancer prevention (Dragsted et al., Pharmacology &Toxicology 1993; 1: 116-135; Craig, American J. Clinical Nutrition 1999;70(3): 491-499; Ray, Indian J. Cancer, 2005; 42(1): 15-24). Severalterpenes have been established as cancer prevention agents in mice.Initiation of breast cancer induced by ionizing radiation or DMBA inrats was almost completely prevented by the monoterpene d-limonene andits hydroxylated derivative, perillyl alcohol, without any evidence oftoxicity, and both agents were effective therapeutically as well(Crowell et al., Carcinogenesis 1992; 13(7): 1261-1264; Gould, J. Cell.Biochem. 1995; 22: 139-144). Geraniol was about 5-fold more effectivethan perillyl alcohol against the growth of human transplantedpancreatic adenocarcinomas (Burke et al., Lipids 1997; 32(2): 151-156).

A triterpene, actein, exhibited a synergistic therapeutic effect with5-fluorouracil (a cancer chemotherapeutic agent) against human breastcancer cells in vitro apparently by causing increased apoptosis (Einbondet al., Planta Medica 2006; 72(13): 1200-1206). The synthetic triterpene2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) at nanomolarconcentrations exhibited potent antiproliferative, pro-differentiation,and anti-inflammatory activities and large increases in thecytoprotective heme oxygenase-1 (HO-1) enzyme in vitro and in vivo. Asimilar inhibition of mouse lung tumors was noted based onanti-oxidative activity. (Liby et al., Cancer Research 2005; 65(11):4789-4798; Liby et al., Cancer Research 2007; 67(6): 2414-2419).Mechanistic studies have found that triterpenes are stronglypro-apoptotic for human small cell lung cancer cells in vitro and act byblocking NF-KappaB thereby elevating apoptosis (Shishodia et al.,Clinical Canc. Res. 2006; 12(6):1828-1838; Kim et al., Molecular CancerTherapeutics 2002; 1(3): 177-184).

Numerous studies have shown major changes in gene expression patternsassociated with terpene exposure (Nakamura et al., FEBS Letters 2004;572(1-3): 245-250; Einbond et al., Anticancer Research 2007; 27(2):697-712; Gould, M. N. Enciron. Health Perspectives 1997; 105: Suppl,977-979; Rahman et al., Cancer Research 2006; 66(9): 4952-4960). Veryfrequently gene expression changes involve clusters of genes directly orindirectly involved in lowering rates of cell proliferation, increasingantioxidative enzymes and increasing apoptosis; all of which tend toblock neoplastic growth. Several key genes that recur often in thesestudies are survivin, NFKappaB, TRAF, bcl-2 and IAP-2 (Shishodia et al.,Clin. Canc. Res. 2006; 12(6): 1828-1838). Colon cancer induced in ratsby 1,2-dimethylhydrazine was inhibited 67% (p<0.01) by 8 mg/kg of thephytoalexin, resveratrol (Sengottuvelan et al., Carcinogenesis 2006;27(5): 1038-1046). Other studies show that hydrophobic receptors mayplay a role in causing apoptosis of cancer cells. Hydrophobic statinsinduced apoptosis and/or growth arrest in HCT116 cells (Powell et al.,Biochem. J. 2001; 356: 481-486) and the cytotoxicity of various organiccompounds against ovarian cancer cells was correlated with theirhydrophobic and steric properties (Verma et al., 2006; 3(4): 441-450).

Hypotheses proposed to explain the cancer preventive activity ofterpenes are generally variations on 1) altered cell signaling throughgene expression changes, 2) antioxidative activity and 3) pro-apoptosiseffects (Niwa et al., Oncology Reports 2007; 17(3): 617-622; Shishodiaet al., 2006; 12(6): 1828-1838; Einbond et al., Anticancer Research2007; 27(2): 697-712; Pusztai et al., Anticancer Research 2007; 27(1A):201-205; Duarte et al., Planta Medica 2006; 72(2): 162-168).Monoterpenes are known to inhibit the isoprenylation of small G proteinswhich affects signal transduction and alters gene expression and canaffect the cell cycle (Gould, Environ. Health Perpsectives 1997; 105:977-979). Studies of mammary cancer show a series of effects includingblockage of the G1 phase of the cell cycle, followed by apoptosis,redifferentiation, and frequently complete tumor regression (Gould,Environ. Health Perpsectives 1997; 105: 977-979; Crowell J. Nutrition1999; 129(3): 775-778). The theme that terpenes can activate apoptosisand can have antioxidative and anti inflammatory activity is recurrentin the literature (Ray, Indian Journal of Cancer 2005; 42(1): 15-24; Weiet al., Carcinogenesis 1993; 14: 1195-1201). For example, quercetin andomega-3-fatty acids in colorectal and prostate cancer responded to theterpene, lycopene (Lambert et al., Am. J. Clin. Nutr. 2005; 81(1):284-291). Others have found that the pyrophosphorylated isoprenoidintermediates and their metabolites are involved in the activation ofthe Vgamma2Vdelta2 T cells (Morita et al., Immun. Reviews 2007; 215:59-76), which enhances the removal of tumor cells because of betterrecognition by T-cells and natural killer receptors. Additionally,hydrophobicity and steric parameters of the terpenes may be one of themost important determinants of cytotoxic activity (Verma et al., Molec.Pharm. 2006; 3(4): 441-450). Some synthetic triterpenes are stronginhibitors of inflammatory processes like induction of nitric oxidesynthase (iNOS) and COX2 but also elevate phase 2 responses, includingheme oxygenase 2 via the Nrf2-Keap1 signaling pathway (Dinkova-Kostovaet al., PNAS 2005; 102(12): 4584-4589).

Atopic dermatitis may be the most prevalent dermatological disease forwhich no fully effective cure currently exists and apparently affectsmany millions worldwide. The cause of atopic dermatitis is unknown, somesuggest that it is a failure of the skin restorative mechanisms, othersinflammation or allergenic reaction. Although it is clear that therestoration of the barrier function of the skin would provide multiplebenefits, including, sunburn protection, reversal of atopic dermatitis,dehydration protection, etc., the skin with atopic dermatitis needsmoisture retention in the epidermal layer. The loss of barrier allowsthe skin to become very dry, which reduces its protective abilities. Inaddition, the skin is very susceptible to recurring disorders, such asstaphylococcal and streptococcal bacterial skin infections, warts,herpes simplex, viral molluscum contagiosum, etc.

Human epidermal differentiation complex (EDC) comprises of several genefamilies: (1) the family of fused genes encoding filaggrin itself andfilaggrin-like proteins (repetin, trichohyalin, cornulin and hornerin),(2) gene cluster with at least of 11 members (loricrin, involucrin and asmall proline-rich region proteins), (3) S100 family genes, with atleast 14 transcripts encoding calcium-binding proteins (11 of them areexpressed in human epidermis). During the physiological formation of thecornified envelope, several genes from each family are expressed in acoordinated way and the epidermal barrier activity is acquired in astepwise manner, firstly with involucrin expression, followed by theexpression of 5100 proteins as well as small proline-rich regionproteins and, finally, loricrin and filaggrin as late differentiationmarkers (Takaishi et al., 2005; J. Biol. Chem. 280:4696-4703).

Abnormal epidermal differentiation is observed in the dermatologicaldiseases. Sagiura et al., 2005; British Journal of Dermatology152:146-149 concluded that a key abnormality in atopic dermatitis mightbe due to the deterioration of epidermal differentiation associated withaltered expression of genes located on chromosome 1q21. Metaphysicalanalysis of public microarray databases for different skin diseasessuggested that there are seven commonly up-regulated genes, DSG3, KRT6,MAP17, PLSCR1, RPM2, SOD2 and SPRR2B. MAP17 is connected with the PDZK1gene where the atopic dermatitis-linked region on human chromosome 1q21is localized. MAP17 is significantly up-regulated in response tointerferon-gamma or interleukin 4 in normal human epidermalkeratinocytes. (Noh et al., 2010; Experimental Dermatology 19(4):355-62)Over-expression of MAP17 in HaCaT keratinocytes significantly decreasedthe expression of filaggrin. It appears that MAP17 is involved in theregulation of the expression of cornified envelope-associated genes atthe 1q21 locus, such as filaggrin, loricrin and involucrin, It appearsthat the T-cell helper cytokine induces the up-regulation of MAP17expression that is linked to the down-regulation of filaggrin in normalhuman epidermal keratinocytes, which may be associated with the abnormalepidermal differentiation observed in the dermatological diseases. (Nohet al., 2010; Experimental Dermatology 19(4):355-62) Theloricrinexpression level was reported to be significantly decreased in chroniclesional atopic dermatitis skin along with deregulated increase in smallproline-rich region proteins (SPRR) 1A and 2C. (Jarzab et al., 2010;International Archives of Allergy & Immunology 151(1):28-37) Latecornified envelope family of genes (e.g. LCE3B and LCE3C) expression canbe induced in normal epidermis by skin barrier disruption and isstrongly expressed in psoriatic lesions, suggesting that compromisedskin barrier function has a role in psoriasis susceptibility. (de Cid etal., 2009; Nature Genetics 41: 211-215)

U.S. Patent Publication 2007/071839 teaches that an inflammatory diseasesuch as atopic dermatitis or acne can be topically treated withcomposition containing terpenes from Ginko Biloba, fluoroglucinols fromHumulus lupulus, and lipophilic compounds enriched in isobutylamides andother natural extracts. Another example of efficacy of monoterpeneperillyl alcohol against the formation and progression ofphotocarcinogenesis indiced by UVB in mice was described by Barthelmanet al., Cancer Res. 1998; 58:711-716. U.S. Patent Publication2010/03052142 A1 by D'Alessio indicates that the topical application ofmonoterpenes was effective to repair tissue as an anti-inflammatoryagent in a variety of skin diseases such as atopic dermitits, seborrheickeratitis, epidermolysis bullosa acquisita, psoriasis, skin alterationsin lupus erythematosus, dermatomyositis, scleroderma, chronic acne,chronic cellulites, pruritus and abnormal or defective scar formation indiabetes. In their example limonene and perillyl alcohol, administeredtopically in a preventive treatment during 3 days at 10 mg/kg/day beforethe induction of skin inflammation, showed a significant effect on skininflammation by reducing the inflammation degree (macroscopic andmicroscopic scores) and by reducing the pro-inflammatory cytokinessecretion (IL-1Φ, IL-6 et TNF-α). Chaudhari (U.S. Patent Publication2009/0036545 A1) teaches that meroterpenes (Sytenol A) act similarly toretinoic acid. Meroterpenes and retinoic acid help with skinrejuvenation, wrinkle control, skin aging through the effect on geneexpression, in particular genes associated with retinoid acid receptors(RAR), dermal-epidermal junction and inflammation e.g. metalloproteinaseMMP28, collagen alpha-6 precursor (COL4A6), heparin sulfotransferase(HS3ST1), lecithin retinol acetyltransferase (LRAT), cytochrome P450,some adherins, cytokeratins, etc.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods of decreasingthe amount of UVR adsorbed by the living cells in the skin byadministering a terpene or a terpenoid. This aspect of the invention maybe alternatively described as a method of inhibiting UVR damage to theDNA of skin cells. In some embodiments, the terpene or terpenoid isadministered orally or topically, in other embodiments, it may beadministered parenterally. The terpene or terpenoid may be, forinstance, a monoterpene, a sesquiterpene or a norisoprenoid, or acombination thereof.

The terpene or terpenoid may be administered 1, 2, 3, 4 or more hoursprior to exposure to UVR, and it may be administered 1, 2, 3, 4, 5, 6,7, or more days, 1 week, 2 weeks, a month, 2 months, 3 months, 6 monthsor more prior to exposure to UVR. Preferably, the terpene or terpenoidis administered within about 2 weeks of exposure to UVR. Likewise, theterpene or terpenoid may be administered 1, 2, 3, 4, 5, or 6 or moretimes per day. In some embodiments the composition contains one or moreof terpenes or terpenoids. In a preferred embodiment, the terpenes orterpenoids utilized are administered in the amounts ranging from about0.6 mg to about 80 mg per kilogram of body weight per day, morepreferably ranging from about 6.0 mg to about 20 mg per kilogram of bodyweight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily. In some embodiments, the terpene or terpenoid is administered inan amount of approximately 0.001% (10 ppm) to 1% (10,000 ppm), and insome instances, the the terpene or terpenoid is administered in the formof a solid or a liquid. In other instances, the terpene or terpenoid isadministered orally or topically. The terpene or terpenoid may in someembodiments be administered orally or topically in an amount of about0.1-1,000 mg, 0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg,5-50 mg, 10-40 mg, or 15-35 mg. In other embodiments, the terpene orterpenoid may be administered in droplets of about 1-1,000 μL, 1-500 μL,1-250 μL, 1-100 μL, 1-50 μL, 1-25 μL, 5-50 μL, 10-40 μL, or 15-35 μL.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to reduce the amount of cutaneousinflammation and erythema present in the skin of subjects receiving theterpene or terpenoid compared to the amount of cutaneous inflammationand erythema present in the skin of subjects not receiving the terpeneor terpenoid. Similarly, the terpene or terpenoid may be administered ina dosage and in amounts sufficient to increase epidermal cell, e.g.keratinocyte, proliferation, cell differentiation, and increase keratinproduction in the skin of subjects receiving the terpene or terpenoidcompared to the amount of epidermal cell proliferation and keratinproduction present in the skin of subjects not receiving the terpene orterpenoid. Similarly, the terpene or terpenoid may be administered in adosage or in amounts sufficient to promote wound healing or to increasethe speed or amount of wound healing. Also, in preferred embodiments theterpene or terpenoid is administered in a dosage and in amountssufficient to alter the production of certain gene products in the skinof subjects receiving the terpene or terpenoid compared to the amount ofproduction of certain gene products present in the skin of subjects notreceiving the terpene or terpenoid. The gene products may be any one ormore of, for instance, NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, proteinkinase C, INOS, PI3-AKT, TNF-A, AP1, NRF2, KEAP1 AND IKappa kinase.Also, in preferred embodiments the terpene or terpenoid is administeredin a dosage and in amounts sufficient to increase the production ofloricrin, filaggrin and late cornified envelope gene products in theskin of subjects receiving the terpene or terpenoid compared to theamount of production of loricrin, filaggrin and late cornified envelopegene products present in the skin of subjects not receiving the terpeneor terpenoid.

In a second aspect, the present invention provides methods ofrejuvenating the skin by administering a terpene or a terpenoid. In someembodiments, the terpene or terpenoid is administered orally, in otherembodiments it may be administered topically or parenterally. Theterpene or terpenoid may be, for instance, a monoterpene, asesquiterpene or a norisoprenoid, or a mixture thereof.

The terpene or terpenoid may be administered 1, 2, 3, 4, 5, or 6 or moretimes per day. In some embodiments terpene is one or more ofbeta-damascenone, nootkatone, or limonene. In some embodiments, theterpene or terpenoid is administered in an amount of approximately0.001% (10 ppm) to 1% (10,000 ppm), and in some instances, the theterpene or terpenoid is administered in the form of a solid or a liquid.In other instances, the terpene or terpenoid is administered orally ortopically. The terpene or terpenoid may in some embodiments beadministered orally or topically in an amount of about 0.1-1,000 mg,0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg, 5-50 mg, 10-40mg, or 15-35 mg. In other embodiments the terpene or terpenoid may beadministered in droplets of about 0.1-1,000 μL, 0.1-500 μL, 0.1-250 μL,0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or 15-35 μL. In apreferred embodiment, the terpenes or terpenoids utilized areadministered in the amounts ranging from about 0.6 mg to about 80 mg perkilogram of body weight per day, more preferably ranging from about 6.0mg to about 20 mg per kilogram of body weight per day. This dosage maybe adjusted to provide the optimum prevention response. For example,several smaller doses may be taken daily.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to reduce the amount of cutaneousinflammation and erythema present in the skin of subjects receiving theterpene or terpenoid compared to the amount of cutaneous inflammationand erythema present in the skin of subjects not receiving the terpeneor terpenoid. Similarly, the terpene or terpenoid may be administered ina dosage and in amounts sufficient to increase epidermal cell, e.g.keratinocyte, proliferation, cell differentiation and increase keratinproduction in the skin of subjects receiving the terpene or terpenoidcompared to the amount of epidermal cell proliferation, epidermal celldifferentiation, and keratin production present in the skin of subjectsnot receiving the terpene or terpenoid. Similarly, the terpene orterpenoid may be administered in a dosage or in amounts sufficient topromote wound healing or to increase the speed or amount of woundhealing. Also, in preferred embodiments the terpene or terpenoid isadministered in a dosage and in amounts sufficient to alter theproduction of certain gene products in the skin of subjects receivingthe terpene or terpenoid compared to the amount of production of certaingene products present in the skin of subjects not receiving the terpeneor terpenoid. The gene products may be any one or more of, for instance,NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, protein kinase C, INOS, PI3-AKT,TNF-A, AP1, NRF2, KEAP1 AND IKappa kinase. Also, in preferredembodiments the terpene or terpenoid is administered in a dosage and inamounts sufficient to increase the production of loricrin, filaggrin, orcaspase 14 gene products in the skin of subjects receiving the terpeneor terpenoid compared to the amount of production of loricrin,filaggrin, or caspase 14 gene products present in the skin of subjectsnot receiving the terpene or terpenoid.

In a third aspect, the present invention provides methods of promotingepidermal cell growth, such as keratinocyte growth, or methods ofincreasing keratin production by administering a terpene or a terpenoid.In some embodiments, the terpene or terpenoid is administered orally ortopically in other embodiments, it may be administered parenterally. Theterpene or terpenoid may be, for instance, a monoterpene, asesquiterpene or a norisoprenoid, or a mixture thereof.

The terpene or terpenoid may be administered 1, 2, 3, 4, 5, or 6 or moretimes per day. In some embodiments terpene is one or more ofbeta-damascenone, nootkatone, or limonene. In a preferred embodiment,the terpenes utilized are administered in the amounts ranging from about0.6 mg to about 80 mg per kilogram of body weight per day, morepreferably ranging from about 6.0 mg to about 20 mg per kilogram of bodyweight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily. In some embodiments, the terpene or terpenoid is administered inan amount of approximately 0.001% (10 ppm) to 1% (10,000 ppm), and insome instances, the the terpene or terpenoid is administered in the formof a solid or a liquid. In other instances, the terpene or terpenoid isadministered orally. The terpene or terpenoid may in some embodiments beadministered orally or topically in an amount of about 0.1-1,000 mg,0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg, 5-50 mg, 10-40mg, or 15-35 mg. In other embodiments, the terpene or terpenoid may beadministered in droplets of about 0.1-1,000 μL, 0.1-500 μL, 0.1-250 μL,0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or 15-35 μL.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to reduce the amount of cutaneousinflammation and erythema present in the skin of subjects receiving theterpene or terpenoid compared to the amount of cutaneous inflammationand erythema present in the skin of subjects not receiving the terpeneor terpenoid. Similarly, the terpene or terpenoid may be administered ina dosage and in amounts sufficient to increase epidermal cellproliferation, epidermal cell differentiation, and increase keratinproduction in the skin of subjects receiving the terpene or terpenoidcompared to the amount of epidermal cell proliferation, epidermal celldifferentiation, and keratin production present in the skin of subjectsnot receiving the terpene or terpenoid. Similarly, the terpene orterpenoid may be administered in a dosage or in amounts sufficient topromote wound healing or to increase the speed or amount of woundhealing. Also, in preferred embodiments the terpene or terpenoid isadministered in a dosage and in amounts sufficient to alter theproduction of certain gene products in the skin of subjects receivingthe terpene or terpenoid compared to the amount of production of certaingene products present in the skin of subjects not receiving the terpeneor terpenoid. The gene products may be any one or more of, for instance,NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, protein kinase C, INOS, PI3-AKT,TNF-A, AP1, NRF2, KEAP1 AND IKappa kinase. Also, in preferredembodiments the terpene or terpenoid is administered in a dosage and inamounts sufficient to increase the production of loricrin, filaggrin, orcaspase 14 gene products in the skin of subjects receiving the terpeneor terpenoid compared to the amount of production of loricrin,filaggrin, or caspase 14 gene products present in the skin of subjectsnot receiving the terpene or terpenoid.

In a fourth aspect, the present invention provides methods of promotingskin wound healing, or methods of increasing the speed or amount ofwound healing, or methods of treating, inhibiting or reducing scarringby administering a terpene or a terpenoid. In some embodiments, theterpene or terpenoid is administered orally or topically in otherembodiments, it may be administered parenterally. The terpene orterpenoid may be, for instance, a monoterpene, a sesquiterpene or anorisoprenoid, or a mixture thereof.

The terpene or terpenoid may be administered 1, 2, 3, 4, 5, or 6 or moretimes per day. In some embodiments terpene is one or more ofbeta-damascenone, nootkatone, or d-limonene. In a preferred embodiment,the terpenes utilized are administered in the amounts ranging from about0.6 mg to about 80 mg per kilogram of body weight per day, morepreferably ranging from about 6.0 mg to about 20 mg per kilogram of bodyweight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily. In some embodiments, the terpene or terpenoid is administered inan amount of approximately 0.001% (10 ppm) to 1% (10,000 ppm), and insome instances, the the terpene or terpenoid is administered in the formof a solid or a liquid. In other instances, the terpene or terpenoid isadministered orally. The terpene or terpenoid may in some embodiments beadministered orally or topically in an amount of about 0.1-1,000 mg,0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg, 5-50 mg, 10-40mg, or 15-35 mg. In other embodiments, the terpene or terpenoid may beadministered in droplets of about 0.1-1,000 μL, 0.1-500 μL, 0.1-250 μL,0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or 15-35 μL.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to reduce the amount of cutaneousinflammation and erythema present in the skin of subjects receiving theterpene or terpenoid compared to the amount of cutaneous inflammationand erythema present in the skin of subjects not receiving the terpeneor terpenoid. Similarly, the terpene or terpenoid may be administered ina dosage and in amounts sufficient to increase epidermal cell, e.g.keratinocyte, proliferation, epidermal cell differentiation, andincrease keratin production in the skin of subjects receiving theterpene or terpenoid compared to the amount of epidermal cellproliferation, epidermal cell differentiation, and keratin productionpresent in the skin of subjects not receiving the terpene or terpenoid.Similarly, the terpene or terpenoid may be administered in a dosage orin amounts sufficient to promote wound healing or to increase the speedor amount of wound healing. Also, in preferred embodiments the terpeneor terpenoid is administered in a dosage and in amounts sufficient toalter the production of certain gene products in the skin of subjectsreceiving the terpene or terpenoid compared to the amount of productionof certain gene products present in the skin of subjects not receivingthe terpene or terpenoid. The gene products may be any one or more of,for instance, NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, protein kinase C,INOS, PI3-AKT, TNF-A, AP1, NRF2, KEAP1 AND IKappa kinase. Also, inpreferred embodiments the terpene or terpenoid is administered in adosage and in amounts sufficient to increase the production of loricrin,filaggrin, or caspase 14 gene products in the skin of subjects receivingthe terpene or terpenoid compared to the amount of production ofloricrin, filaggrin, or caspase 14 gene products present in the skin ofsubjects not receiving the terpene or terpenoid.

In a fifth aspect, the present invention provides compositions usefulfor one or more of increasing the amount of UVR that the skin can blockor absorb, for inhibiting UVR damage to the skin, for rejuvenating theskin, for promoting epidermal cell growth, such as keratinocyte growth,or increasing keratin production, or for promoting wound healing, orincreasing the speed or amount of wound healing containing one or moreterpene or terpenoid. The terpene or terpenoid may be, for instance, amonoterpene, a sesquiterpene or a norisoprenoid. In a preferredembodiment, the terpenes are provided in amounts sufficient to beadministered in amounts ranging from about 0.6 mg to about 80 mg perkilogram of body weight per day, more preferably ranging from about 6.0mg to about 20 mg per kilogram of body weight per day. This dosage maybe adjusted to provide the optimum prevention response. For example,several smaller doses may be taken daily. The terpene or terpenoid mayin some embodiments be administered orally or topically in an amount ofabout 0.1-1,000 mg, 0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg,0.1-25 mg, 5-50 mg, 10-40 mg, or 15-35 mg. In other embodiments, theterpene or terpenoid may be provided in a droplet in an amount of about0.1-1,000 μL, 0.1-500 μL, 0.1-250 μL, 0.1-100 μL, 0.1-50 μL, 0.1-25 μL,5-50 μL, 10-40 μL, or 15-35 μL. The composition containing the terpeneor terpenoid may further contain other ingredients, such as, forinstance a pharmaceutically acceptable carrier. The composition may be aliquid or a solid, such as for instance a tablet, lozenge or pill, andmay be administered orally, topically or parenterally.

In a sixth aspect, the present invention provides a kit containing oneor more compositions useful for one or more of increasing the amount ofUVR that the skin can block or absorb, for inhibiting UVR damage to theskin, for rejuvenating the skin, for promoting epidermal cell growth,such as keratinocyte growth, or increasing keratin production, or forpromoting wound healing, or increasing the speed or amount of woundhealing. The compositions contain one or more terpene or terpenoid. Theterpene or terpenoid may be, for instance, a monoterpene, asesquiterpene or a norisoprenoid, or a mixture thereof. In a preferredembodiment, the terpenes are provided in amounts sufficient to beadministered in the amounts ranging from about 0.6 mg to about 80 mg perkilogram of body weight per day, more preferably ranging from about 6.0mg to about 20 mg per kilogram of body weight per day. This dosage maybe adjusted to provide the optimum prevention response. For example,several smaller doses may be taken daily. The terpene or terpenoid mayin some embodiments be administered orally or topically in an amount ofabout 0.1-1,000 mg, 0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg,0.1-25 mg, 5-50 mg, 10-40 mg, or 15-35 mg. In other embodiments, theterpene or terpenoid may be provided in a droplet in an amount of about0.1-1,000 μL, 0.1-500 μL, 0.1-250 μL, 0.1-100 μL, 0.1-50 μL, 0.1-25 μL,5-50 μL, 10-40 μL, or 15-35 μL. The composition containing the terpeneor terpenoid may further contain other ingredients, such as, forinstance a pharmaceutically acceptable carrier. The composition may be aliquid or a solid, such as for instance a tablet, lozenge or pill, andmay be administered orally, topically or parenterally. The kit furthercontains instructions for administering, providing or taking thecomposition.

In a seventh aspect, the present invention provides methods of restoringthe barrier function of the skin by administering a terpene or aterpenoid. This aspect of the invention may be alternatively describedas a method of increasing the expression of genes associated with thestratum granulosum or stratum corneum or both, decreasing or eliminatingthe number or size of breaches in the stratum granulosum and stratumcorneum, or decreasing the amount of moisture or fluid that may passthrough the skin in a given period of time. In some embodiments, theterpene or terpenoid is administered orally or topically, in otherembodiments, it may be administered parenterally. The terpene orterpenoid may be, for instance, a monoterpene, a sesquiterpene or anorisoprenoid, or a mixture thereof.

The terpene or terpenoid may be administered 1, 2, 3, 4, 5, 6, 7, ormore days, 1 week, 2 weeks, a month, 2 months, 3 months, 6 months ormore. Preferably, the terpene or terpenoid is administered daily.Likewise, the terpene or terpenoid may be administered 1, 2, 3, 4, 5, or6 or more times per day. In some embodiments the composition containsone or more of terpenes or terpenoids. In a preferred embodiment, theterpenes or terpenoids utilized are administered in the amounts rangingfrom about 0.6 mg to about 80 mg per kilogram of body weight per day,more preferably ranging from about 6.0 mg to about 20 mg per kilogram ofbody weight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily. In some embodiments, the terpene or terpenoid is administered inan amount of approximately 0.001% (10 ppm) to 1% (10,000 ppm), and insome instances, the the terpene or terpenoid is administered in the formof a solid or a liquid. In other instances, the terpene or terpenoid isadministered orally or topically. The terpene or terpenoid may in someembodiments be administered orally or topically in an amount of about0.1-1,000 mg, 0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg,5-50 mg, 10-40 mg, or 15-35 mg. In other embodiments, the terpene orterpenoid may be administered in droplets of about 0.1-1,000 μL, 0.1-500μL, 0.1-250 μL, 0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or15-35 μL.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to significantly restore the stratumgranulosum and stratum corneum, by, for example, upregulation of thegenes for filaggrin, loricrin, caspase 14, hornerin and other latecornified envelope components which results in increasing thickness ofthe stratum corneum, decreasing or eliminating the number or size ofbreaches in the stratum granulosum and stratum corneum, or increasingthe amount of hydrophobic amino acids or decreasing the amount ofmoisture or fluid that may pass through the skin in a given period oftime. Similarly, the terpene or terpenoid may be administered in adosage and in amounts sufficient to increase epidermal cell, e.g.keratinocyte, proliferation, cell differentiation and increase keratinproduction in the skin of subjects receiving the terpene or terpenoidcompared to the amount of epidermal cell proliferation and keratinproduction present in the skin of subjects not receiving the terpene orterpenoid. Similarly, the terpene or terpenoid may be administered in adosage or in amounts sufficient to promote wound healing or to increasethe speed or amount of wound healing. Also, in preferred embodiments theterpene or terpenoid is administered in a dosage and in amountssufficient to alter the production of certain gene products in the skinof subjects receiving the terpene or terpenoid compared to the amount ofproduction of certain gene products present in the skin of subjects notreceiving the terpene or terpenoid. The gene products may be any one ormore of, for instance, NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, proteinkinase C, INOS, PI3-AKT, TNF-A, AP1, NRF2, KEAP1 AND IKappa kinase.Also, in preferred embodiments the terpene or terpenoid is administeredin a dosage and in amounts sufficient to increase the production offilaggrin, loricrin, caspase 14, hornerin and other late cornifiedenvelope components gene products in the skin of subjects receiving theterpene or terpenoid compared to the amount of production of filaggrin,loricrin, caspase 14, hornerin and other late cornified envelopecomponents gene products present in the skin of subjects not receivingthe terpene or terpenoid.

In an eighth aspect, the present invention provides methods of treatinga dermatological disease, such as, for instance atopic dermatitis,eczema, dry skin, cracked skin, canker sores, and acne by administeringa terpene or a terpenoid. The methods of treating may feature preventingor inhibiting the development or progress of the dermatological disease,such as, for instance atopic dermatitis, eczema, dry skin, cracked skin,canker sores, and acne. The terpene or terpenoid may be, for instance, amonoterpene, a sesquiterpene or a norisoprenoid, or a mixture thereof.This aspect of the invention may include providing amounts sufficient tosignificantly restore the stratum granulosum and stratum corneum, by,for example, upregulation of the genes for filaggrin, loricrin, caspase14, hornerin and other late cornified envelope components which resultsin increasing thickness of the stratum corneum, decreasing oreliminating the number or size of breaches in the stratum granulosum andstratum corneum, or increasing the amount of hydrophobic amino acids ordecreasing the amount of moisture or fluid that may pass through theskin in a given period of time. In some embodiments, the terpene orterpenoid is administered orally or topically, in other embodiments, itmay be administered parenterally.

The terpene or terpenoid may be administered 1, 2, 3, 4, 5, 6, 7, ormore days, 1 week, 2 weeks, a month, 2 months, 3 months, 6 months ormore. Preferably, the terpene or terpenoid is administered daily.Likewise, the terpene or terpenoid may be administered 1, 2, 3, 4, 5, or6 or more times per day. In some embodiments the composition containsone or more of terpenes or terpenoids. In a preferred embodiment, theterpenes or terpenoids utilized are administered in the amounts rangingfrom about 0.6 mg to about 80 mg per kilogram of body weight per day,more preferably ranging from about 6.0 mg to about 20 mg per kilogram ofbody weight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily. In some embodiments, the terpene or terpenoid is administered inan amount of approximately 0.001% (10 ppm) to 1% (10,000 ppm), and insome instances, the the terpene or terpenoid is administered in the formof a solid or a liquid. In other instances, the terpene or terpenoid isadministered orally or topically. The terpene or terpenoid may in someembodiments be administered orally or topically in an amount of about0.1-1,000 mg, 0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg,5-50 mg, 10-40 mg, or 15-35 mg. In other embodiments, the terpene orterpenoid may be administered in droplets of about 0.1-1,000 μL, 0.1-500μL, 0.1-250 μL, 0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or15-35 μL.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to significantly reduce one or moresymptoms associated with a dermatological disease, such as, forinstance, atopic dermatitis, eczema, dry skin, cracked skin, cankersore, and acne. Also, in some embodiments, the terpene or terpenoid isadministered in a dosage and in amounts sufficient to significantlyrestore the stratum corneum, stratum granulosum by, for example,upregulation of the genes for filaggrin, loricrin, caspase 14, hornerinand other late cornified envelope components which results in increasingthe thickness of the stratum corneum, decreasing or eliminating thenumber or size of breaches in the stratum granulosum and stratumcorneum, or increasing the amount of hydrophobic amino acids ordecreasing the amount of moisture or fluid that may pass through theskin in a given period of time. Similarly, the terpene or terpenoid maybe administered in a dosage and in amounts sufficient to increaseepidermal cell proliferation, cell differentiation and increase keratinproduction in the skin of subjects receiving the terpene or terpenoidcompared to the amount of epidermal cell, e.g. keratinocyte,proliferation, cell differentiation and keratin production present inthe skin of subjects not receiving the terpene or terpenoid. Similarly,the terpene or terpenoid may be administered in a dosage or in amountssufficient to promote wound healing or to increase the speed or amountof wound healing. Also, in preferred embodiments the terpene orterpenoid may be administered in a dosage and in amounts sufficient toalter the production of certain gene products in the skin of subjectsreceiving the terpene or terpenoid compared to the amount of productionof certain gene products present in the skin of subjects not receivingthe terpene or terpenoid. The gene products may be any one or more of,for instance, NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, protein kinase C,INOS, PI3-AKT, TNF-A, AP1, NRF2, KEAP1 AND IKappa kinase. Also, inpreferred embodiments the terpene or terpenoid is administered in adosage and in amounts sufficient to increase the production of loricrin,filaggrin and late cornified cell envelope gene products in the skin ofsubjects receiving the terpene or terpenoid compared to the amount ofproduction of loricrin, filaggrin and late cornified envelope geneproducts present in the skin of subjects not receiving the terpene orterpenoid.

In a ninth aspect, the present invention provides methods of increasingthe thickness of the stratum corneum by administering a terpene orterpenoid. In some embodiments, the terpene or terpenoid is administeredorally or topically, in other embodiments, it may be administeredparenterally. The terpene or terpenoid may be, for instance, amonoterpene, a sesquiterpene or a norisoprenoid, or a mixture thereof.

The terpene or terpenoid may be administered 1, 2, 3, 4, 5, 6, 7, ormore days, 1 week, 2 weeks, a month, 2 months, 3 months, 6 months ormore. Preferably, the terpene or terpenoid is administered daily.Likewise, the terpene or terpenoid may be administered 1, 2, 3, 4, 5, or6 or more times per day. In some embodiments the composition containsone or more of terpenes or terpenoids. In a preferred embodiment, theterpenes or terpenoids utilized are administered in the amounts rangingfrom about 0.6 mg to about 80 mg per kilogram of body weight per day,more preferably ranging from about 6.0 mg to about 20 mg per kilogram ofbody weight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily. In some embodiments, the terpene or terpenoid is administered inan amount of approximately 0.001% (10 ppm) to 1% (10,000 ppm), and insome instances, the the terpene or terpenoid is administered in the formof a solid or a liquid. In other instances, the terpene or terpenoid isadministered orally or topically. The terpene or terpenoid may in someembodiments be administered orally or topically in an amount of about0.1-1,000 mg, 0.1-500 mg, 0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg,5-50 mg, 10-40 mg, or 15-35 mg. In other embodiments, the terpene orterpenoid may be administered in droplets of about 0.1-1,000 μL, 0.1-500μL, 0.1-250 μL, 0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or15-35 μL.

In preferred embodiments, the terpene or terpenoid is administered in adosage and in amounts sufficient to increase the thickness of thestratum corneum by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or morecompared to the thickness of the stratum corneum before the terpene orterpenoid is first administered. Also, in some embodiments, the terpeneor terpenoid is administered in a dosage and in amounts sufficient tosignificantly restore the stratum corneum, stratum granulosum by, forexample, upregulation of the genes for filaggrin, loricrin, caspase 14,hornerin and other late cornified envelope components which results inincreasing the thickness of the stratum corneum, decreasing oreliminating the number or size of breaches in the stratum granulosum andstratum corneum, or increasing the amount of hydrophobic amino acids ordecreasing the amount of moisture or fluid that may pass through theskin in a given period of time. Similarly, the terpene or terpenoid maybe administered in a dosage and in amounts sufficient to increaseepidermal cell proliferation, cell differentiation and increase keratinproduction in the skin of subjects receiving the terpene or terpenoidcompared to the amount of epidermal cell proliferation, celldifferentiation and keratin production present in the skin of subjectsnot receiving the terpene or terpenoid. Similarly, the terpene orterpenoid may be administered in a dosage or in amounts sufficient topromote wound healing or to increase the speed or amount of woundhealing. Also, in preferred embodiments the terpene or terpenoid may beadministered in a dosage and in amounts sufficient to alter theproduction of certain gene products in the skin of subjects receivingthe terpene or terpenoid compared to the amount of production of certaingene products present in the skin of subjects not receiving the terpeneor terpenoid. The gene products may be any one or more of, for instance,NFKappaB, COX1, COX2, MAPKS, PGE2, ODC, protein kinase C, INOS, PI3-AKT,TNF-A, AN, NRF2, KEAP1 AND IKappa kinase. Also, in preferred embodimentsthe terpene or terpenoid is administered in a dosage and in amountssufficient to increase the production of loricrin, filaggrin and latecornified cell envelope gene products in the skin of subjects receivingthe terpene or terpenoid compared to the amount of production ofloricrin, filaggrin and late cornified envelope gene products present inthe skin of subjects not receiving the terpene or terpenoid.

In a tenth aspect, the present invention provides methods of generatingor stimulating growth of skin such as artificial skin by providing oradministering a terpene or terpenoid. In some instances the skin may bemammalian skin, and in some instances the skin may be human skin.

The terpene or terpenoid may be provided or administered 1, 2, 3, 4, 5,6, 7, or more days, 1 week, 2 weeks, a month, 2 months, 3 months, 6months or more. Preferably, the terpene or terpenoid is provided oradministered daily. Likewise, the terpene or terpenoid may be providedor administered 1, 2, 3, 4, 5, or 6 or more times per day. The terpeneor terpenoid may be, for instance, a monoterpene, a sesquiterpene or anorisoprenoid, or a mixture thereof. In some embodiments the compositioncontains one or more terpenes or terpenoids. In some embodiments, theterpene or terpenoid is provided or administered in an amount ofapproximately 0.001% (10 ppm) to 1% (10,000 ppm), and in some instances,the the terpene or terpenoid is provided or administered in the form ofa solid or a liquid. The terpene or terpenoid may in some embodiments beprovided or administered in an amount of about 0.1-1,000 mg, 0.1-500 mg,0.1-250 mg, 0.1-100 mg, 0.1-50 mg, 0.1-25 mg, 5-50 mg, 10-40 mg, or15-35 mg. In other embodiments, the terpene or terpenoid may be providedor administered in droplets of about 0.1-1,000 μL, 0.1-500 μL, 0.1-250μL, 0.1-100 μL, 0.1-50 μL, 0.1-25 μL, 5-50 μL, 10-40 μL, or 15-35 μL.

In preferred embodiments the terpene or terpenoid may be provided oradministered in a dosage and in amounts sufficient to alter theproduction of certain gene products in the skin. The gene products maybe any one or more of, for instance, NFKappaB, COX1, COX2, MAPKS, PGE2,ODC, protein kinase C, INOS, PI3-AKT, TNF-A, AP1, NRF2, KEAP1 AND IKappakinase. Also, in preferred embodiments the terpene or terpenoid isprovided or administered in a dosage and in amounts sufficient toincrease the production of loricrin, filaggrin and late cornified cellenvelope gene products.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows 253 2-fold unregulated genes sorted into categories. Onlythe category of epidermal cell differentiation (red color) wassignificantly altered in comparison to random. The loricrin gene wasincluded in this category.

FIG. 2 shows 341 2-fold down-regulated genes sorted into genecategories. The significantly altered categories in comparison to randomare shown in red and include many stress-response type of categories.

FIG. 3 demonstrates a protective effect of ingested terpenes against thesunburn effect of solar-spectrum UVR in the skin of SKH1 hairless mice(FIG. 3A) and shows that the terpenes stimulate epidermal hyperplasiaand increase the thickness of the keratin layer (FIG. 3B). The cutaneousinflammation and erythema associated with a 15 minute exposure ofhairless mouse skin to 3.0 kJ/m2 of UVR was blocked in mice that werefed 4 daily 30 microL droplets of 3 different terpenes (d-limonene,nootkatone and beta-damascenone) per os.

FIG. 4 demonstrates that the stratum corneum was consistently thickenedby the action of the β-damascenone. The increased stratum corneumthickness may not be correlated with increased thickness of thenucleated keratinocyte population, which generally is believed to be thesource of the complex structural features of the stratum corneum.

FIG. 5 demonstrates that the stratum corneum was consistently thickenedby the action of the β-damascenone. The increased stratum corneumthickness may not be correlated with increased thickness of thenucleated keratinocyte population, which generally is believed to be thesource of the complex structural features of the stratum corneum.

FIG. 6 provides a graph showing how different β-damascenone treatmentpatterns affected the statistical significance of the increasedthickness. The horizontal line indicates the boundary betweensignificance (above the line) and lack of significance (below the line).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides for reversing deficiencies in the stratumcorneum or restoring the stratum corneum, the ‘barrier’ layer of skin,which leads to at least two important benefits including increasedfiltering of UV radiation which leads to sunburn protection and reduceddehydration (loss of moisture) which reduces or eliminates the diseaseprocess of atopic dermatitis. Thereby, the present invention provides ameans for treating, preventing or curing dermatological diseases such asatopic dermatitis. Some ways in which the present invention providessuch means is by upregulation of the genes for filaggrin, loricrin,caspase 14, hornerin and other late cornified envelope components whichresults in increasing thickness of the stratum corneum, decreasing oreliminating the number or size of breaches in the stratum granulosum andstratum corneum, or increasing the amount of hydrophobic amino acids ordecreasing the amount of moisture or fluid that may pass through theskin in a given period of time.

By “reversing deficiencies in the stratum corneum” or “restoring thestratum corneum” is meant, for example, increasing the thickness and thecomposition of the stratum granulosum, stratum corneum, decreasing oreliminating the number or size of breaches in the stratum corneum, ordecreasing the amount of moisture or fluid that may pass through theskin in a given period of time. The thickness of the stratum corneum maybe increased by about, for instance, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, two times, three times, or more. Similarly, the number ofbreaches in the stratum corneum by may be reduced by about, forinstance, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.Likewise, the cumulative or the average size of such breaches may bereduced by about, for instance, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or more.

The term “dermatological disease” is intended to include all diseasesinvolving the skin or pathologies of the skin that involve all or inpart or are caused all or in part by deficiencies of the stratumgranulosum and stratum corneum. Stated differently, the term“dermatological disease” is intended to include all diseases involvingthe skin or pathologies of the skin that may be treated by or whosesymptoms may be improved by “reversing deficiencies in the stratumcorneum” or “restoring the stratum corneum.” Examples of suchdermatological diseases include atopic dermatitis, seborrheic keratitis,epidermolysis bullosa acquisita, psoriasis, skin alterations in lupuserythematosus, dermatomyositis, scleroderma, acne, cellulites, pruritusand scar formation in diabetes.

The present invention is based in part upon results from experimentsusing animal models demonstrating that ingestion of terpenes such asbeta-damascenone, d-limonene and nootkatone results in UVR-protection,increased keratinocyte formation, upregulation of loricrin and filaggrinand vascular endothelial growth factor (VEGF) genes, and downregulationof matrix metalloproteinases. The present invention is based in partupon the discovery that terpenes and terpenoids promote UVR-absorbinghyperplasia and/or hyper keratinization. The terms “terpene” and“terpenoid” are defined very broadly herein. The terms “terpene” and“terpenoid” are meant to include all compounds that comprisehydrocarbons containing one or more of an isoprene unit[CH₂═C(CH₃)—CH═CH₂ or (C₅H₈).]. The terpenoid may be one of the acyclicterpenoids, cyclic terpenoids, cycloaliphatic compounds that arestructurally related to terpenoids, and mixtures thereof. The terpenoidsinclude terpenes and terpene derivatives such as, for instance,alcohols, aldehydes, acetals, ketones, acids, esters, and terpenecompounds that contain heteroatoms such as nitrogen or sulfur, andmixtures thereof. Further, the terpenoids include such compounds asMyrcene, Ocimene, beta-FameSene, Dihydromyrcenol, Geraniol, Nerol,Linalool, Myrcenol, Lavandulol, Citronellol, Trans-trans-Farnesol,Trans-Nerolidol, Citral, Citral diethyl acetal, Citronellal,Citronellyloxyacetaldehyde, 2,6,10-Trimethyl-9-undecenal, Tagetone,Solanone, Geranylacetone, Cis-Geranic acid Citronellic acid, Geranylformate, Geranyl acetate, Geranyl propionate, Geranyl isobutyrate,Geranyl isovalerate, Neryl acetate, Lynalyl formate, Linalyl acetate,Linalyl propionate, Linalyl butyrate, Linalyl isobutyrate, Lavendulylacetate, Citronellyl formate, Citronellyl acetate, Citronellylpropionate, Citronellyl isobutyrate, Citronellyl isovalerate,Citronellyl tiglate, Cis-Geranic acid nitrile, Citronellic acid nitrile,and mixtures thereof. Still further, the terpenoid may be one ofLimonene, Alpha-Terpinene, Gamma-Terpinene, Terpinolene,Alpha-Phellandrene, Beta-Phellandrene, Alpha-Pinene, Beta-Pinene,Camphene, 3-Carene, Caryophytlene, (+)-Valencene, Thujopsene,Alpha-Cedrene, Beta-Cedrene, Longifolene, (+)-Neoiso-isopulegol,Isopulegol, Alpha-Terpineol, Beta-Terpineol, Gamma-Terpineol,Delta-Terpineol, 1-Terpinen-4-ol, Carvone, Alpha-Ionone, Beta-Ionone,Gamma-Ionone, Alpha-Irone, beta-Irone, gamma-Irone,alpha-n-Methylionone, beta-n-Methylionone, gamma-n-Methylionone,alpha-Isomethylionone, beta-Isomethylionone, gamma-Isomethylionone,Allylionone, Pseudojonone, n-Methylpseudoionone, Isomethylpseudoionone,Damascones, beta-Damascenone, Nootkatone, Cedryl methyl ketone,Alpha-Terpinyl acetate, Nopyl acetate, Khusymil acetate, and mixturesthereof. In addition, the terpenoid may be a cycloaliphatic compoundstructurally related to terpenoids such as5-(2,2,3-Trimethyl-3-cyclopenten-1-yl)-3-methylpentan-2-ol,2,4-Dimethyl-3-cyclohexenecarboxaldehyde, 4-(4-Methyl-3-penten-1-yl)-3-cyclohexene carboxaldehyde,4-(4-Hydroxy-4-methypentyl)-3-cyclohexene carboxaldehyde, Civetone,Dihydrojasmone, Cis-Jasmone, 5-Cyclohexadecen-1-one,2,3,8,8-Tetramethyl-1,2,3,4,5,6,7,8-octahydro-2-napthalenyl methylketone, 3-methyl-2-cyclopenten-2-ol-1-one,4,7-Methano-3a,4,5,6,7,7a-hexahydro-5-(or 6)-indenyl acetate, Allyl3-cyclohexylpropionate, Methyl dihydrojasmonatemethyl(3-oxo-2-pentylcyclopentyl)acetate, and mixtures thereof.

These data demonstrate that terpenes and terpenoids provide a protectiveeffect against UVR damage to the skin of hairless mice. The skininflammation associated with exposure of hairless mouse skin to UVR wasblocked in mice that were fed 3 different terpenes (d-limonene,nootkatone and beta-damascenone). On the contrary, daily exposures toterpenes following UVR exposure failed to produce any protective effect,indicating that anti-oxidation is not a likely explanation of theprotective effect when the terpenes precede the UVR exposure.Histopathology results demonstrate well that terpenes triggerhyperplasia of the skin. Terpenes and terpenoids provide a three tofive-fold increase in keratinocytes demonstrating that the terpenesalter the skin response prior to exposure to the UVR.

Terpenes may suppress cancer induction by activating genes that produceUVR-absorbent keratin thereby reducing photodamage to DNA or byinactivating inflammation-relevant genes that otherwise would elevateoxidative DNA damage. Diet is proven to exert major influences on cancerincidence in human populations (Lu et al. Experimental Biology &Medicine 2007; 232(4): 473-480; Wu et al. PNAS 2004; 101(18):7094-7099;Borek, C. Integrative Canc. Therapies 2004; 3(4): 333-341; Li et al.,Toxicology Letters 2003; 144(3): 397-406). Migrants often acquire thecancer risks of their adopted land much more quickly than could beexpected based on any known natural selection process (Ziegler et al.,Journal of the National Cancer Institute 1993; 85: 1819-1827). In labanimals, work is currently focused on elucidating the underlyingmechanisms of cancer chemoprevention for a variety of preventivecompounds (Weinstein, Cancer Research, 1988; 48(15): 4135-4143; Dragstedet al., Pharmacology & Toxicology 1993; 72: 116-135; Steele et al.,Mutation Research 2003; 523-524: 137-144; Waladkhani et al., Int. J.Molec. Med 1998; 1(4): 747-753). Terpenes are a family of compoundscommonly found in the vital juices of plants. They include some of themost potent chemopreventive agents known, e.g. lycopene and perillylalcohol, and generally have been proven safe as dietary additives. Thepossibility cannot be discounted that a portion of the anti-carcinogeniceffect of vegetable-based diets is based on the terpene content.

Two separate types of DNA damage, direct and oxidative, in epithelialcell DNA following the same exposure protocols for UVR and terpenes havebeen quantified. Cyclobutyl pyrimidine dimers (CPDs) and 8-oxo-dG areoften utilized as a measure of direct UVR-induced DNA damage andoxidative DNA damage respectively. It may be that terpene-induced UVRabsorption is the basis for sunburn and cancer preventive activity ofthe terpenes.

An important clue to the mechanism of the terpene effect was obtainedfrom histological evaluation of skin samples from the preliminaryexperiment. Terpenes stimulated epidermal cell proliferation and keratinproduction. The combination of a thicker epidermis and keratin layer mayhave acted as a shield by absorbing some portion of the UVR. Such apossibility was reinforced by the observation that the increasedepidermal thickness (hyperplasia) was more pronounced in the mice thatreceived both, terpene and UVR, which raises the possibility that theresponse may be a protective adaptation to UVR. A literature searchturned up nothing related to proliferative stimulation of epithelialcells by terpenes in skin or any other organ. Some reports show anopposite effect, i.e. terpenes block proliferation and induce apoptosisin several organs, but there were no reports of such activity in skin.Histopathological diagnosis was performed on the skin samples.Histopathology results demonstrated remarkably well, that the terpenestriggered the hyperplasia of the epidermis. Three to five-foldadditional increases in keratinocyte layers demonstrates that theterpenes alter the skin response to the UVR. One can speculate thateither a metabolic product or the terpenes themselves could trigger theincreased proliferation of the epidermal basal cells.

The significance of these preliminary findings is that terpenes could beinitiating a new type of sunblock, the mechanism of which is currentlyunknown but could be part of an adaptation that is analogous to thewell-known UVR-blocking effect of melanin.

By “effective amount” is meant such amount that is capable of performingthe function of the compound or property for which an effective amountis expressed. As known to those skilled in art, the exact amountrequired may vary from case to case, depending on recognized variablessuch as, for example, the compounds employed and the individual subjecttreated. Thus, it is often not desirable to specify an exact “effectiveamount.” However, an appropriate effective amount may be determined byone of ordinary skill in the art using only routine experimentation.

It will be apparent to those skilled in the art that it is not intendedthat the invention be limited by the examples, and that themodifications can be made without departing from the invention, asdefined by the appended claims. It is intended that all modificationsand variations be included with the scope of the invention. The claimsare meant to cover the claimed components and steps in any sequence thatis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

Example 1

A pharmaceutical or dietary supplement form may be made by conventionalprocedures known in the industry that is by mixing active compounds withedible acceptable solid or liquid carriers or excipients in any formcapsules, tablets, dragees, saches, films, powders, lozenges, syrups,liquid suspensions, emulsions and solutions in a convenient dosage form.Three different formulations of terpenes were made for oral usage andtwo formulations for topical application. Effective amount of terpene isneeded to provide protection against UVR. Generally, an effective amountof the oral formulation is taken per day for the duration of 1-7 daysprior to the UVR, or more preferably 3-4 days, in association with acarrier, and particularly one in which the active ingredients aresoluble per se or are effectively solubilized (e.g. as an emulsion ormicroemulsion). The terpenes may be administered in the amounts rangingfrom about 0.6 mg to about 80 mg per kilogram of body weight per day,more preferably ranging from about 6.0 mg to about 20 mg per kilogram ofbody weight per day. This dosage may be adjusted to provide the optimumprevention response. For example, several smaller doses may be takendaily.

Formulation I Terpene 50% by weight (0.4 g/capsule) Sunflower oil 50% byweight (0.4 g/capsule)Capsules are prepared by dispersing active ingredient terpene in theliponate oil or any other oil such as sunflower, canola, etc. oils. Theoil can be used as is or can be filled into soft, elastic gelatincapsules.

Formulation II Terpene 50% by weight (0.4 g/tablet or capsule)Microcrystalline cellulose 25% by weight (0.2 g/tablet or capsule)Lactose BP 25% by weight (0.2 g/tablet or capsule) Ethyl cellulose 0.1 gfor coating of a tabletTerpenes, microcrystalline cellulose, and lactose BP are mixed, andextruded which is followed by spheronisation of the extrudate anddrying. The dried pellets are then coated with release-controllingmembrane (ethyl cellulose) and filled into a two-piece, hard gelatincapsule.

According to the invention, the formulations are administered topically(transdermally) when terpenes are applied in various carriers such aslubricants, wetting agents, emulsifying and suspending agents,preservatives, antiirritants, emulsion stabilizers, film formers, gelformers, odor masking agents, resins, hydrocolloids, solvents,solubilizers, neutralizing agents, permeation accelerators, pigments,quaternary ammonium compounds, refatting and superfatting agents,ointments, cream or oil base materials, silicone derivatives,stabilizers, sterilizing agents, propellants, drying agents, opacifiers,thickeners, waxes, emollients, or white oils. Suitable carriers can bewater, alcohols, oils which can dissolve or disperse active ingredient.

It is contemplated that in addition to the active ingredient terpene oneor more antioxidants are also used.

The active ingredient may be administered preferably once a day for theduration of 1-7 days, more preferably between 3-4 days prior to the UVR.An effective amount of terpene is needed to provide protection againstUVR. Generally, an effective amount of the topical formulation isapplied to exposed skin sites in association with a carrier, andparticularly one in which the active ingredients are soluble per se orare effectively solubilized (e.g. as an emulsion or microemulsion).Preferably, the formulation contains 0.05 to 50% by weight of terpene.Desirably, it will contain 0.1%-20% by weight, or more preferably 0.2-5%by weight of terpene.

Formulation III Terpene  1% by weight (1 g) Liponeate oil 99% by weight(99 g)The terpene component is mixed with the oil part, mixed well together toensure complete solution. The mixture is used topically as such for 3-4days prior to the UV radiation.

Formulation IV Terpene  1% by weight (1 g) Cream base 99% by weight (99g)The active ingredient terpene is mixed with other components of thecream together to ensure complete distribution of terpene. The mixtureis applied topically as such for 3-4 days prior to the UVR.

Experimental Design

1. experiment with various concentrations

2. experiment with delayed exposure

3. experiment with topical application, 100%

4. experiment with various topical concentrations

All experiments were performed with female hairless mice (SKH1)utilizing solar spectrum ultraviolet radiation (UVR). Each experimentwas performed in triplicate including controls and UVR-exposed mice andincluded a total of 22 mice per experiment for a total of 66 mice. Theprotocol included untreated mice, UVR-irradiated mice as controls forthe terpene treatments, UVR+terpene mice and terpene-only mice.

Four solar simulating FS-20 fluorescent lamps were used to deliver 3kJ/m² measured by a calibrated International Light UV radiometer at thelamp distance between 20 to 24 cm. The UVR dose was selected to be justabove the minimal erythemal dose for producing a ‘sunburn’ reaction inmouse skin (this is very similar to the minimal erythemal dose offair-skinned humans).

Three terpenes, d-limonene, nootkatone and beta-damascenone, were testedfor their sunburn prevention potency. Each terpene was given to eachmouse per os (by mouth) in 15-30 μL droplets (˜15-30 μg) viamicropipette once each day for 4 consecutive days prior to and/orsubsequent to UVR exposure. Animals were observed and photographed every24 hours for a period of 6-7 days after the irradiation. Each terpenewas administered in pure form.

Terpene d-limonene was also tested for its sunburn prevention potencywhen UVR delivered with a delay. Pure d-limonene was given to mice peros in the amount of 20 μl per day for four (4) days. The UVR exposure of3 kJ/m² was delivered seven (7) days after the last dose wasadministered. The animals were observed and photographed. No erythemawas observed. Terpene d-limonene was also tested topically to preventsunburn. Pure or diluted d-limonene, 20 μl was spread on the dorsal partof mice for four (4) days prior to the UVR exposure of 3 kJ/m².Liponeate, a triglyceride fraction of coconut oil was used as a diluent.Concentration 1, 10 and 100% were tested. Development of erythema wasobserved and photographed after 6-7 days.

Synergy of terpenes was tested by applying a mixture of three differentterpenes that were mixed in equal proportion. The mixture (20 μl) wasapplied on the dorsal part of mice for four (4) days prior to the UVRexposure of 3 kJ/m2. Development of erythema was observed andphotographed after 6-7 days. The efficacy of the mixture was comparedagainst individual terpenes at the same concentration.

All three terpenes exhibited complete sunburn protection when givenprior to the UVR, but were inactive given after the UVR. The histologyshowed that the terpenes stimulated epidermal hyperplasia (piling upindicating proliferation).

Animals for the histology and microarray analysis were euthanized 24hours after 4 days of terpene pre-feed and the UVR exposure. Tissuesamples from skin and other organs were frozen and prepared forhistological diagnosis and for microarray analysis, which consisted oftesting gene expression by utilizing Affymetrix mouse microarray chipscontaining about 39,000 transcripts with 45,101 probe sets. Individualgenes found to be induced by the UVR were predominantlyinflammation-related and included NFKappaB, COX1, COX2, MAPKs, PGE2,ODC, protein kinase C, iNOS, PI3-Akt, TNF, AN, Nrf2, Keap1 and IKappakinase. Significant reductions in the constellation ofinflammation-related genes was noted in the mice exposed to theterpenes, which is consistent with the protective action of the terpenesagainst sunburn, which is basically an inflammatory reaction to the UVRexposure.

Gene expression microarray analysis was performed as follows: RNA wasextracted from a 0.1 g sample of mouse skin and an equal quantity ofkeratinocytes by using the TRIZOL reagent (Invitrogen, Carlsbad, Calif.,USA) according to the manufacture's instructions and 200 μg of total RNAwas used for the isolation of poly A+mRNA by using Oligotex mRNA minicolumns (QIAGEN, Valencia, Calif., USA). The final centrifugation wasperformed after the incubation with 1/10 volume of 3.0 M NaOAc, 2.5volumes of ethanol and 1 μl glycogen (Boehringer Mannheim GmbH,Mannheim, Germany) at −20° C. overnight. Then the pellet was resuspendedin diethylpyrocarbonate (DEPC)-treated water. RNA was labeled withbiotin and hybridized to a Mouse genome 430_2 GeneChip® microarray(Affymetrix, Inc. Santa Clara, Calif., USA). Data from the Affymetrixmouse expression array chip were imported into the BRB-ArrayToolsVersion 3.3.0 developed by the Biometric Research Branch of the U.S.National Cancer Institute for normalization and analysis. Statisticalcomparison between the treated group and control group based on threereplicates was performed by using the class comparison tool of BRB ArrayTool software, which uses two sample T-tests (with random variancemodel) to find genes whose expression varied significantly. A value ofP≤0.001 was chosen as statistically significant. In order to identifysignificantly altered categories of genes, the Gene Ontogeny website wasutilized as documented in the GOTree Machine software suite. A list ofsignificantly altered genes derived from the BRB-ArrayTools software wasuploaded into the GOTree software for derivation of significantlyaltered gene categories. For these calculations the entire list of geneson the Affymetrix (mouse 430_2) microarray was used as the referencelist.

Mice were treated with either UVR or UVR+beta-damascenone, and samplesof tissue were removed from the dorsal skin for RNA extraction andanalysis on an Affymetrix Microarray System utilizing mouse whole genomearray chips Affy 430_2. The UVR sample served as the control in thispreliminary experiment. The results for the combined treatment areexpressed relative to the UVR response. Graph 2A shows that the onlysignificantly (p<0.05) altered category at level 4 of the Gene OntologyTree was “epidermal cell differentiation” which is consistent with thehistological observations that the keratinizing layer in the epidermiswas greatly increased by the terpene in comparison to UVR. Graph 2Bshows the results for the down regulated genes. Here 13 categories weresignificantly (p<0.05) down-regulated in the terpene+UVR treatmentrelative to the UVR only. These down regulations can be attributed tothe action of the terpene and include a total of 13 categories of which5 are strongly associated with the inflammatory reaction, namely: immunesystem process, inflammatory response, immune response, physiologicaldefense response, and response to wounding. In other wordsinflammation-relevant genes were strongly down-regulated. Thesignificance of these down-regulations with respect to cancer inductionis not clear, but they are consistent with suppression of the sunburnreaction by the terpene.

Example 2

Research Design

Experiments will be performed to establish in SKH1 hairless mouse skinhow gene expression alterations based on custom expression microarrays,sunburn severity and cancer induction depend on the quantity of the 3selected terpenes. Preliminary experiments indicated that the terpenes,d-limonene, nootkatone and beta-damascenone given prior to UVR as purecompounds per os in 4 daily 30 microL droplets blocked UVR-inducedsunburn. Protocols will be carried out at lowerper os terpeneconcentrations for establishing a dose-response in regard to geneexpression alterations, sunburn prevention and cancer prevention.

Table 1 indicates the basic protocol for concentrations of terpenes anddoses of UVR to provide baseline information on the changes in geneexpression in the SKH1 hairless mouse skin. Adequate levels (80%) ofstatistical power require generally at least 3 replicates of eachexposure group as indicated in Table 1. Practically however it is morecost-efficient to conduct experiments utilizing only 1 chip per 1 mouse.More chips and mice might be needed in special cases where responses arelower, e.g. as the terpenes are diluted. Additionally it may not benecessary to conduct the full protocol as outlined in Table 1 for all 3terpenes, as the preliminary study indicated the differences betweenthem are small. Where needed, additional replications will providebetter estimates of average responses and lower p-values for testing thestatistical significance of any differences that may occur.

TABLE 1 Outline of custom microarray experiments - number of mice Geneexpression in mouse skin pretreated daily with terpenes for 4 days priorto UVR exposure. Tissue samples to be taken 24 h after the UVR exposure.UVR exposures will be 15 min of solar simulating FS-20 fluorescent lamps(3.0 kJ/m²) Column # 1 2 3 4 5 6 7 8 Dilution Control UVR Limo. Limo. +UVR Noot. Noot. + UVR Dama. Dama. + UVR Total 10% 3 3 3 3 3 3 3 3 24 50%3 3 3 3 3 3 3 3 24 100% 3 3 3 3 3 3 3 3 24 Note: Limo. = d-limonene,Noot. = nootkatone, Dama. = beta-damascenone *Solvent will be theinactive triglyceride, liponate

Based on a combination of sunburn suppression and gene expressionalterations, a carcinogenesis experiment will be setup to quantify thedegree of cancer prevention that can be expected for a given amount ofsunburn suppression and/or gene expression alteration. Thecarcinogenesis experiment of necessity will be carried out with chronicUVR (0.8 kJ/m² 3× weekly

TABLE 2 Carcinogenesis protocol - number of hairless mice Undilutedd-limonene will be administered once per os 30 μL at times prior to UVirradiation. UVR exposures will be 15 min of solar simulation FS-20fluorescent lamps (3.0 kJ/m2) Terpene Start Time prior to UVR Limon. +UVR (d) Control only Limon. UVR Noot. Noot. + UVR Damas. Damas. + UVR 43 3 3 3 3 3 3 3 6 3 3 3 3 3 3 3 3 10 3 3 3 3 3 3 3 3 14 3 3 3 3 3 3 3 3Total mice 96 Note: Limon. = d-limonene, Noot. = Nootkatone, Damas. =beta-damascenonefor 26 weeks) along with a d-limonene supplement (concentration to bedetermined in relation to gene expression alterations) in the lab chow.Selection of a terpene concentration will be based on custom microarrayresponses to lab chow supplement with differing concentrations ofd-limonene. All experiments will be conducted with skin but tissue fromother organs will be preserved for possible future analysis.

The carcinogenesis protocol is designed to include 2 concentrations ofd-limonene (not yet determined) and one group of UVR only. Each groupwill contain 30 mice and continue for 26 weeks.

A custom mouse Affymetrix microarray chip containing ˜150 keratin- andinflammation-relevant genes identified in the preliminary study will beutilized routinely. Examples of individual genes of special interestare: loricrin, NFKappaB, COX1, COX2, MAPKs, PGE2, ODC, protein kinase C,iNOS, PI3-Akt, TNF, AP1, Nrf2, Keap1 and IKappa kinase. Whole genomemouse microarrays containing 39,000 transcripts with 45,101 probe setswill be utilized selectively in addition to the custom arrays to insureconsistent responses at lower terpene concentrations or different entrypoints. The custom chips are a cost-efficient way of revealing how genecategories associated with the suspected terpene mechanism of actionvary with terpene concentration.

An initial experiment will be carried out with d-limonene (by a smallmargin the most potent pure inhibitor of the 3 terpenes) applied as inthe preliminary experiment, i.e. 30 μL per os 4 times daily followed byUVR 24 h after the final terpene dose. Tissue sampling for microarrayanalysis will be performed 24 h after the UVR. At 1 chip per 1 mouse,the per os dilution protocol for d-limonene will require 9 chips and 9mice (columns 1, 2 and 3 in Table 1. An analogous topical applicationprotocol will require an additional 9 chips and 9 mice. These initialexperiments will provide sufficient data to draw conclusions concerninghow the gene expression alterations depend on d-limonene concentrationand entry mode. An additional 18 mice would be used to evaluate forsunburn suppression at the same d-limonene concentrations. The sameprotocol will be carried out for beta-damascenone as a second terpene toestablish whether potency differences exist between terpenes at lowerconcentrations. Another experiment will be carried out to determine ifthe 4 day pre-exposure provides optimal protection. This will involveextending the length of the pre-exposure from 4 d to 10 d and 14 d toestablish whether additional protective benefit is available inreference to sunburn prevention (Table 2). If the hypothesis is truethat the protective response involves a piling up of keratinocytes andkeratin, the signal must occur considerably in advance of the pointwhere the protection becomes effective, because of the need for cellproliferation and the formation of keratin and probably loricrin.Epidermal cell cycle times are generally about 10 days in controlepidermis but can be shortened to about 1.5 to 2 days. Still a number ofdays would be needed to accumulate the cell numbers and keratin massnoted on the histological preparations in the preliminary studies.

Cancer yields are calculated by assigning a number and at death oreuthanasia after which histological sections are prepared and evaluatedfor each tumor. Only histologically verified lesions are scored asmalignant neoplasms and assigned to histological type. Availablesoftware evaluates cancer yields interactively and projects the responseto a pre-chosen standard time point or until a preselected standarddeviation is reached. Groups are terminated when standard deviations gobelow 20% (total tumors=25) of the mean response; a strategy thatminimizes caging time and animal per diem fees without compromisingprecision or conclusions. Based on linear additivity and a Poissondistribution, the cancer yield standard deviations are in the range of15% to 25%. Accordingly a 50% or greater response difference betweengroups of 25 animals is detectible with a p-value=0.05 and type 2error=0.20.

Sample size and statistical analysis by assigning animals toexperimental and control groups based on the assumption that at least a20% difference in tumor yields between control and treatment groups willbe obtained. Several factors are considered in the power analysis: (i)the minimum true effect of a treatment, which is regarded assignificant; (ii) the standard deviation or random error variance, S2;(iii) the significance level of the statistical test, which is set at aP-value=0.05; and (iv) the power of detecting the true effect of atreatment, P, commonly 0.8. For an expected minimum effect of 20% and astandard deviation of 15%, a minimum sample size of 16 per treatmentgroups is required to achieve a type 2 error <15%. ANOVA will be used tocompare the effect of radiations on body weights in wild-type versusBubR1+/− mice. ANOVA is robust against departure from normality, butnon-parametric tests will be utilized as necessary. Additivity will betested by using the statistical hypothesis that the expected number oftumors per rat is invariant under fractionation of the total dose. Thepreliminary data for a single fraction of 3 Gy gives an average of 5cancers per 20 rats. This yields λ0=0.25 per rat as the tumor rate. Thealternate hypotheses λ1 is chosen for a pairwise comparison to be|λ1=λ0±½λ0, so that the absolute difference between alternatives is|2−λ1|=0.125. The Poisson assumption implies a population standarddeviation of σ=(λ0)½=0.125 so that the normalized difference is|λ1−λ0|/σ=1.0. Consequently the per-group sample size formula for ourproblem reduces to N=2(Z1−σ/2+Z1−β)2 where the Z's are percentiles ofthe standard normal distribution corresponding to the significance level{acute over (σ)} and power 1−β. Because 5 pairwise comparisons will bemade, the desired 0.05 significance level is to be adjusted for theindividual comparisons by the Bonferroni method, i.e. to use {acute over(α)}=0.05/5=0.01, and similarly the desired power of 0.80 is to beachieved by using β=(1−0.8)/5=0.05. Thus Z1−σ/2=Z0.995=2.576 andZ1−β=Z095=1.645. Putting this all together the result is that N=36animals per group.

Results

The data will provide gene expression levels in treated skin incomparison to controls for the terpenes alone and for the UVR alone andthen for the various combinations at a time prior to onset of a visiblesunburn reaction. These data should provide an immediate indicationwhether the terpenes produce a consistent change in gene expressionpatterns and how much the UVR response differs from or is altered byterpenes as concentration changes. In similar experiments in rat skinexposed to ionizing radiation, it was shown that vitamin A, while havingno effect on gene expression in controls, blocked about 80% of theinflammation-relevant gene expression alterations caused by the x-rays(Lu et al., Experimental Biology & Medicine 2007 232(4): 473-480). Inthe referenced work the principal effect of the radiation was toactivate inflammation-relevant genes in immunocytes that invaded theskin. Something similar is expected in these proposed experiments,although the response to UVR might differ from that of ionizingradiation. Gene expression studies were carried out for d-limonene at 1hr before the UVR exposure and 1 hour after UVR exposure; the latterbeing roughly the peak of the inflammatory response in these mice.

Gene expression microarray results will provide insight into how theterpenes are working, because the whole genome mouse chip contains all39,000 transcripts with 45,101 probe sets in the Affymetrix microarrayanalysis system. The whole genome chip provides a scan of the entiregenome and possibly reveals gene categories associated with all possiblesuspected mechanisms of action, including proliferative, anti-oxidativeand apoptosis genes, etc. In addition the whole genome chips providesinformation on categories that are not yet suspected of involvement incancer prevention, as well as, information on responses of individualgenes, such as, NFKappaB, COX1, COX2, MAPKs, PGE2, ODC, protein kinaseC, iNOS, PI3-Akt, TNF-α, AP1, Nrf2, Keap1 and IKappa kinase. Theimportant point here is that these scans permit identification ofcategories of genes and even possibly individual genes as putativebiomarkers in tumor prevention. Once such genes are identified by thegenome chips, custom arrays will be fabricated and utilized at muchlower expense. The data on comparative oxidative stress levels asquantified by 8-oxo-dG levels and the incidence of apoptosis quantifiedby the TUNEL assay will be available and should provide confirmation forthe gene expression responses to some extent. Certainly establishing anyone of these endpoints as putative tumor prevention markers would be apositive contribution from these studies.

Likely, the terpenes will bind to the cell membrane receptors without athreshold, i.e. the relationship between terpene concentration andsunburn or cancer prevention should monotonically increase with terpeneconcentration possibly plateauing at the higher levels as a possiblescenario. Several studies have shown that terpenes are capable ofcausing direct cellular responses, such as apoptosis induction andanti-oxidation in vitro which supports the idea that terpenes caninteract directly with cells. Possibly 2 mechanisms are actingindependently, one based on systemic responses and one based on a directinteraction between epithelial cell and terpene. It is well-establishedthat TPA, a terpene, acts in vitro by binding directly to protein kinaseC (Wu et al., Proceedings of the National Academy of Sciences of theUnited States of America 2004; 101(18):7094-7099; Borek, IntegrativeCancer Therapies 2004; 3(4): 333-341).

Methods and Procedures:

UV Irradiation Technique

Mice will be irradiated with a bank of four parallel Westinghousesolar-spectrum FS-20 fluorescent lamps either once at a dose of 3.0kJ/m² for short term protocols or 3 times per week at a dose of 1.0kJ/m² for long-term carcinogenesis protocols. The UVR dose rates will bemeasured with a calibrated IL 1400A digital radiometer/photometerequipped with a SEL240 UVB-1 detector (International Light, Inc.). TheUVR fluence is 0.2 kJ/m²/min and 1.0 kJ/m² is approximately 30% of theminimal erythemic dose (MED) for these mice.

Diets

Initially terpenes will be administered per os via pipette. If neededfor final testing, cereal-based diets will be formulated with equivalentterpene concentrations and supplied as a powder containing designatedterpenes prepared by Dyets, Inc., Bethlehem Pa. Chemicals will bepurchased from Sigma Biochemical, St. Louis, Mo. The experimentalconcentrations of terpenes are expected to be non-toxic to mice.Terpenes were a gift from Biokeys for Flavors, LLC, Norwood, N.J.

Gene Expression Microarray Analysis for Keratinocytes and Skin Samples

RNA will be extracted from a 0.1 g sample of mouse skin and an equalquantity of keratinocytes by using the TRIZOL reagent (Invitrogen,Carlsbad, Calif., USA) according to the manufacture's instructions and200 μg of total RNA will be used for the isolation of poly A+mRNA byusing Oligotex mRNA mini columns (QIAGEN, Valencia, Calif., USA). Thefinal centrifugation will be performed after the incubation with 1/10volume of 3.0 M NaOAc, 2.5 volumes of ethanol and 1 μl glycogen(Boehringer Mannheim GmbH, Mannheim, Germany) at −20° C. overnight. Thenthe pellet will be resuspended in diethylpyrocarbonate (DEPC)-treatedwater. RNA will be labeled with biotin and hybridized to a Mouse genome430_2 GeneChip® microarray (Affymetrix, Inc. Santa Clara, Calif., USA).Data from the Affymetrix mouse expression array chip are imported intothe BRB-ArrayTools Version 3.3.0 developed by the Biometric ResearchBranch of the U.S. National Cancer Institute for normalization andanalysis. Statistical comparison between the treated group and controlgroup based on 3 replicates each was performed by using the classcomparison tool of BRB Array Tool software, which uses two sampleT-tests (with random variance model) to find genes whose expressionvaried significantly, a value of P≤0.001 was chosen as statisticallysignificant. In order to identify significantly altered categories ofgenes, the Gene Ontogeny website will be utilized as documented in theGOTree Machine software suite. A list of significantly altered genesderived from the BRB-ArrayTools software will be uploaded into theGOTree software for derivation of significantly altered gene categories.For these calculations the entire list of genes on the Affymetrix (mouse430-2) microarray will be used as the reference list.

Example 3

Experiments will be performed to quantify in SKH1 mouse skin 2 separatetypes of DNA damage, direct and oxidative, in epithelial cell DNAfollowing the same exposure protocols for UVR and terpenes as describedin Aim #1. Cyclobutyl pyrimidine dimers (CPDs) and 8-oxo-dG will beutilized as a measure of direct UVR-induced DNA damage and oxidative DNAdamage respectively. With the results from Aim #1, these data will provewhether or not terpene-induced UVR absorption is the basis for sunburnand cancer preventive activity.

These experiments are based on the endpoint of induction of cyclobutylpyrimidine dimers (CPDs) in the DNA of skin epithelial cells by UVR.Preliminary experiments have shown that inflammation genes induced byUVR can be blocked by each of 3 terpenes, d-limonene, nootkatone andbeta-damascenone, administered per os daily for 4 days in 30 microLdroplets prior to irradiation. Female SKH1 hairless mice will be exposedto the solar spectrum UVR for 15 minutes at a distance of 20 cm from abank of 4 FS-20 fluorescent lamps. Typically inflammation reaches a peakbetween 3-6 days after the UVR exposure.

TABLE 3 Protocol for CPDs, 6, 4 PPs, 8-oxo-dG and TUNEL assays - numberof mice Mouse skin pretreated daily with a terpene for 4 days prior toUVR exposure. Tissue samples to be taken 24 h after the UVR exposure.UVR dorsal skin exposure: 15 min of solar simulating FS-20 fluorescentlamps (3.0 kJ/m²) Column # 1 2 3 4 5 6 7 8 Dilution* Control UVR LimoLimo. + UVR Noot. Noot. + UVR Dama. Dama. + UVR Total 1/10 3 3 3 3 3 3 33 24 ½ 3 3 3 3 3 3 3 3 24 1/1 3 3 3 3 3 3 3 3 24 Note: Limo. =d-limonene, Noot. = nootkatone, Dama. = beta-damascenone *Solvent willbe the inactive triglyceride, liponate

Oxidative stress levels in the form of 8-oxo-dG in the skin epitheliumwill be measured every other day for 6 days after UVR exposure eitherwith or without supplementary terpenes prior and subsequent to the UVR.The protocol is designed to establish at what approximate concentrationeach terpene loses its effectiveness as a sunburn retardant. PCNAprocedures will be carried out on skin tissues obtained in theseexperiments to confirm that proliferative responses are occurring asexpected. Occasional tunel assays will be carried out to monitorapoptosis during and after the various exposures. These results will beimportant for confirming the timing of gene expression via microarrayanalysis might be needed to confirm when local gene expression signalsare altered in relation to the administration of the terpenes. Theseexperiments are designed for assaying all 3 terpenes, but d-limonenewill be tested initially. The other terpenes might be tested later forconfirmatory purposes.

Initial experiments will involve d-limonene. Each of the followingassays will be carried out on skin tissue obtained from 4 differentlocations on each mouse. The assays are: CPDs, PCNA, 8-oxo-dG and TUNEL.The 6.4 photoproduct assay will on occasion be carried out as an adjunctto the CPD assay, because they are somewhat redundant although theirrepair rates in mouse skin differ markedly. The photolesions will be anobjective measure of the amount of UVR that has penetrated to thecutaneous epithelium. Selective localization of the photoproducts willbe pursued by using an in situ procedure an approach to identifyingepithelial cells with the greatest cancer risk for comparison withcarcinogenesis studies. The 8-oxo-dG assay will provide information toverify that inhibition of inflammation-relevant genes by the terpeneproduces measurable reductions of localized oxidative stress as assumed.

The results of experiments will provide exact 1:1 comparisons. Tissuesampling will be performed 24 h after the UVR. At 4 assays per 1 mouse,the per os dilution protocol will require 36 assays on 9 mice. Ananalogous topical application protocol will require an additional 9mice. These initial experiments could possibly provide sufficient datato draw conclusions concerning how the gene expression alterations arerelated to the various objective endpoints for d-limonene. The sameprotocol will be carried out for beta-damascenone as a second terpene toestablish whether potency differences might exist between terpenes atlower concentrations and whether comparisons with gene expressionresults are consistent or at variance.

Results

The CPDs with backup from 6.4 PPs will establish the comparative amountof UVR getting to the cutaneous epithelial cells in relation to dose andtype of terpene. However as both assays are immune-based andimmune-based methods can vary, redundancy should be accepted as anadvantage until proven otherwise. Recent studies of the carcinogenicenhancement of UVR in mouse skin by arsenic in the drinking waterindicates that the combination of DNA photodamage by the UVR andoxidative DNA damage in the form of 8-oxo-dG by the arsenic causesnearly a 5-fold synergistic increase in the yield of squamouscarcinomas; a co-carcinogenic effect. If so even a small reduction ofthe oxidative stress by the terpene might produce a substantialreduction in the yield of cancer, if sunburn and cancer are as closelylinked as is generally believed. Because other documented effects ofterpenes includes increased proliferations and apoptosis, the PCNA andTUNEL assays will be performed selectively as a backup to thepossibility that oxidative stress levels may not be a consistentbiomarker of sunburn and/or skin cancer inhibition by the terpenes.

The prevention of sunburn by the terpene will induce epidermaldifferentiation (keratinization) genes that absorb the UVR which will beapparent in the results as a reduction in the CPDs and/or 6.4 PPs. Inaddition the custom microarrays will confirm that inflammation geneswere down-regulated by the terpene and that will be quantified as areduction in the level of 8-oxo-dG in the epithelial DNA. In this waygene expression changes would be verified independently, and it mighteven be possible to make use of carcinogenesis data to predict thedegree of cancer prevention to be expected from a given combination oflower DNA photodamage and lower oxidative stress levels.

Methods and Procedures

Oxidative DNA Damage in Skin of Mice (8-Oxo-dG)

Mouse DNA will be extracted from 100 mg or larger samples of tissue byusing the DNA isolation kit from Roche Diagnostics Corporation(Indianapolis, Ind.). The DNA will be dissolved in AE elution buffer(Qiagen, Valencia, Calif.), and 100 μg will be digested as describedpreviously. The following incubations will be performed: DNase I for 30min at 37° C.; NP1 for 60 min at 37° C.; AP for 30 min at 37° C.; andphosphodiesterase inhibitors PDEI and PDEII for 30 min at 37° C. Theincubation mixture will be filtered through a 0.5 ultrafree centrifugalfilter (Millipore, Billerica, Mass.). The iron chelator DTPA will beadded to the DNA hydrolysates to prevent artificial oxidation. Twenty μlof hydrolysate will be analyzed by high-performance liquidchromatography. The high-performance liquid chromatography with electroncapture detection system consisted of an Agilent 1100 binary pump,autosampler, and variable wavelength detector controlled by ChemstationSoftware 7.01 (Agilent Technologies, Palo Alto, Calif.); an ESACoulochem II electrochemical detector (ESA, Inc., Chelmsford, Mass.); aC18 Alltima guard column, 7.5×4.6 mm, particle size of 5 μm (Alltech,Deerfield, Ill.); and a YMC ODS-AQ column, 4.6×15 cm, 120 Å, S-5 (WatersCorporation, Milford, Mass.). The mobile phase consisted of 8% aqueousmethanol containing 50 mM sodium acetate buffer (pH 5.2). Elution willbe isocratic at a flow rate of 0.8 ml/min. The deoxyguanosineconcentration will be monitored based on absorbance (245 nm), and8-oxo-dG concentration will be based on the electrochemical reading (400mV). Levels will be quantified using the standard curves of eachcompound. The degree of DNA damage will be expressed as 8-oxo-dG per 10⁶deoxyguanosine.

Quantitation of UV Photoproducts by Me-Linked Immunosorbent Assay(ELISA)

After UV-irradiation or after subsequent DNA repair, genomic DNA ispurified from the keratinoctes isolated from mouse epidermis and isheat-denatured. Polyvinylchloride flat-bottom microtiter plates(Dynatech, Alexandria, Va.) precoated with 1% protamine sulfate (Sigma)is then incubated with mouse genomic DNA at 37° C. for 20 hour. Afterdrying, the plates are washed 5 times with PBS containing 0.05% Tween 20(PBS-T). The plates are then incubated withl % goat serum in PBS (150μl/well) at 37° C. for 1 hr to prevent non-specific binding of theantibody and washed again. 100 μl of the 6.4 photoproduct or cyclobutylpyrimidine dimer (CPD) antibody (generously provided by Dr. Toshio Mori)are added to each of 3 wells and incubated at 37° C. for 30 min. Theplates are washed 5 times with PBS-T and then incubated with 100 μl of abiotin-F(ab)₂ fragment goat anti mouse IgG(H+L) ( 1/2000 in PBS, Zymed)at 37° C. for 30 min. The plates are washed 5 times with PBS-T and thenincubated with 100 μl of streptavidin conjugated with peroxidase (1/10000 in PBS, Zymed) at 37° C. for 20 min. After 3 washings with PBS-Tand 2 subsequent washings with citrate-phosphate buffer (pH 5.0), 100 μlof substrate solution consisting of 0.04% o-phenylene diamine and 0.007%H₂O₂ in citrate-phosphate buffer is added to each well. Following 30 minincubation at 37° C., 50 μl of 2M H₂SO₄ is added to stop the reaction,and absorbance at 492 nm is measured by using an HTS 7000 Bio AssayReader (Perkin Elmer). Standard curves are obtained from DNA samplesextracted immediately after UV irradiation.

In Situ Visualization of CPD and PCNA

Two monoclonal antibodies, TDM-2 for CPD (and 64-M2 for 6.4 PPs, ifneeded), will be used for visualizing of UV induced DNA damage. Thespecificity of these antibodies has been examined in detail, nodetectable crossreactivity was found between CPD and 6.4 PPs. Skinbiopsies cut as 4-04 thick cryosections are fixed with 0.001%paraformaldehyde for 3 min at 4° C. and dehydrated through a gradedmethanol series. Sections are microwaved (500 W) for a total of 5 min in10 mM citrate buffer, pH 6.0, and are then treated with 0.1% trypsin for30 min at room temperature. Slides are subsequently treated with 0.07 MNaOH in 70% ethanol for 15 min at room temperature to denature DNA.After blocking and RNase A treatment, slides are incubated with TDM-2 at1:10,000 dilution or with 64M-2 at 1:1,500 dilution with 5% fetal bovineserum at 4° C. overnight. The binding of monoclonal antibodies torespective photolesions in each nucleus can be detected with abiotin-streptavidin system and FITC. The nuclei are then counterstainedwith propidium iodide. For double staining of CPD and PCNA, a PCNAmonoclonal antibody (Clone 24, Transduction Laboratory, Lexington, Ky.)at 1:50 dilution is used and denaturation of DNA is omitted. The dishesare washed with phosphate-buffered saline, mounted in drops of ProLongAntifade (Molecular Probes, Eugene, Oreg.), and coverslipped.Fluorescent images are obtained by using an InSIGHTplus-IQlaser-scanning confocal microscope (Meridian Instruments, Okemos,Mich.).

Assay of DNA Fragments as Indicator of Apoptosis (TUNEL Assay)

The TUNEL (terminal deoxynucleotide transferase {TdT}-mediateddigoxigenin-uridine triphosphate {dig-UTP} nick-end labeling whichmeasures 3′-hydroxyl ends of DNA fragments formed during programmed celldeath, i.e. apoptosis. Apoptotic cells are detected using the DeadEndColorimetric TUNEL system (Promega, Madison, Wis.) followingmanufacturer's protocol with some modifications. The tissue is fixed in1% paraformaldehyde, then sectioned at 6 microns and processed accordingto the Apop Tag-Fluorescein (Oncor, Inc., Gaithersburg, Md.) protocol(55). Briefly the slides are incubated first with TdT and dig-UTP andthen with fluorescein isothiocyanate (FITC)-labeled anti-digoxigeninantibody (anti-dig-FITC). The slides are then washed with Triton X-100and counterstained with 5 ug/ml propidium iodide. Cells are thenobserved with a fluorescent microscope and measurements are made of therelative green or red fluorescence by using the appropriate band passfilters and a photometer. About 100 cells will be quantified toestablish the average ratio. Sections are mounted after dehydration andobserved under 400× magnification for TUNEL-positive cells.

Female hairless (SKH1) mice will be utilized in the proposed work for 2purposes: 1) to establish comparative gene expression patterns andcancer induction in skin for different doses of per os or topicalexposure to 3 terpenes (d-limonene, nootkatone, and beta-damascenon)prior to UVR and 2) for evaluating the comparative yields of direct DNAdamage (CPDs and 6.4 photoproducts) versus oxidative DNA damage(8-oxo-dG) in mouse skin exposed to terpenes and UVR as in 1). The workaddresses the question whether the cancer preventive properties of theterpenes depend on whole body exposure or direct chemical interactionsin skin. Approximately 190 mice will be needed over the full studyincluding mice to establish the effect of the terpenes on geneexpression alterations, anti-oxidative levels and apoptosis incidence.Where required lab chow diets will be formulated and supplied as apowder containing designated terpenes at specified concentrations byDyets, Inc., Bethlehem Pa. Chemicals will be purchased from SigmaBiochemical, St. Louis, Mo. The proposed concentrations and entry routesof terpenes are not expected to produce toxicity in SKH1 mice.

These experiments require the induction of malignant (squamouscarcinomas) and benign (papillomas) neoplastic lesions in the skin ofSKH1 mice. No in vitro system or theoretical approach is currentlyavailable that could provide reliable information as a surrogate fortumor induction in animals. The anti-carcinogenic effectiveness ofterpenes acting through direct or indirect pathways is a poorlyunderstood phenomenon and findings from in vitro studies would not bepersuasive in comparison to equivalent results obtained in animals.

Veterinary care is provided by the School of Medicine animal facility. Aveterinarian visits the facility once per month on a routine basis andmore frequently if special needs arise. The facility was within the lastyear partially converted from conventional to a barrier facility, butthis study will not use the barrier. The animals will be checked dailyfor overt signs of illness and mortality. Each batch of new animals willbe quarantined for 8 weeks, during which time they are observed forsigns of overt illness and abnormalities. Sentinel mice are periodicallyculled and complete serological, bacteriological, parasitological andpathological examinations are performed by animal care personnel. Onlyif the sentinels are completely free of disease and abnormalities willexperimentation commence on the quarantined animals.

It is not expected that any procedures to be employed in the proposedexperiments will produce pain or distress to the mice not relieved byappropriate analgesia or anesthesia. The use of animals is essential forthese studies, since the protocols involve cancer induction. The animalswill have ad libitum access to food and water. Fresh food is suppliedtwice weekly, and excreta are removed twice weekly. The racks and cagesare cleaned every two weeks. Cages will be placed on wheeled racks inconventional, well ventilated (12-15 changes per hour) animal room keptat 22+/−2° C. and 55+/−15% relative humidity, and lighted on a 12hour/day timer. The animals will be checked daily for overt signs ofillness and mortality. Moribund animals will be euthanized by CO₂asphyxia. The cereal-based diet adequate in all essential nutrients willbe purchased from Purina Inc. Mice will be fed 3 times per week.Sentinel mice kept with each experiment are examined periodically and atthe termination of the experiment, when complete serological,bacteriological, parasitological and pathological examinations areperformed. The N.Y.U. Medical Center Animal Facilities are registeredwith and approved by the US Department of Agriculture (21-118). TheI.E.M. Animal Facility and all experimental protocols are subject toapproval of the N.Y.U. Medical Center IACUC, while animal care andmaintenance, including housing, are conforming to the NIH Guide for theCare and Use of Laboratory, and the facility is AALAC accredited.

The method of euthanasia will be i.p. injection with 120 mg/kg of bodyweight of sodium pentobarbital or CO₂ inhalation depending on the assaybeing performed. Both methods are approved by the Panel on Euthanasia ofthe American Veterinary Medical Association.

Example 4

The present experiment will be performed for evaluating theeffectiveness and persistence of sunburn protection afforded bydifferent doses and exposure regimens of a test terpene, β-damascenone.Female SKH-1 strain mice, obtained from Charles River Breeding Labs,Wilmington, Mass., will be fed per os various amounts of the terpene asindicated in the following Tables 4 and 5.

TABLE 4 Group Dose/ # of Total # Application Applications Dose SampleTime* # of mice 1 20 uL 4 daily 80 uL 1 day 3 2 10 uL 4 daily 40 uL 1day 3 3 80 uL 1 only 80 uL 1 day 3 4 20 uL 1 only 20 uL 1 day 3 5  0 uL— 0 5 day 3 6 20 uL 4 daily 80 uL 5 day 3 7 10 uL 4 daily 40 uL 5 day 38 80 uL 1 only 80 uL 5 day 3 9 20 uL 1 only 20 uL 5 day 3 10 20 uL 4daily 80 uL 12 day  3 11 10 uL 4 daily 40 uL 12 day  3 12 80 uL 1 only80 uL 12 day  3 13 20 uL 1 only 20 uL 12 day  3 14 Total 39 *The sampletime will be measured from the time of the final exposure.

TABLE 5 Results of β-damascenone-induced sunburn protection study inhairless mice Areas Treatment with Areas with Total area P-value(one-tailed) Group UVR Damas. sunburn no sunburn (mice) ValueSignificance 1 1.5 kJ/m² None 8.0 4.0 12.0 (3) — — 2 1.5 kJ/m² 20 uL × 41.0 11.0 12.0 (3) 0.003 high 3 1.5 kJ/m² 10 uL × 4 2.0 6.0  8.0 (2)0.068 borderline 4 1.5 kJ/m² 20 uL × 4 0.0 8.0  8.0 (2) 0.003 high 5 1.5kJ/m² 10 uL × 1 5.0 7.0 12.0 (3) 0.219 not significant 6 1.5 kJ/m² 20 uL× 1 2.0 10.0 12.0 (3) 0.013 high 7 1.5 kJ/m² 40 uL × 1 1.0 3.0  4.0 (1)0.146 not significant 2 + 4 1.5 kJ/m² 20 uL × 4 1.0 19.0 20.0 (5) 0.001very high Note 1: Damas. = β-damascenone; mice were fed indicatedamounts of Damas by feeding tube either 4 times daily (×4) of 1 time(×1). Ultraviolet radiation was applied to a 2.0 × 5.0 cm area of dorsalmouse skin at 5 days after the final Damas application and sunburn wasevaluated 5 days later. Note 2: Sunburn was evaluated in 4 equal,contiguous subregions (1.25 cm × 2.0 cm) running anterior to posteriorwithin the UVR exposed area of each mouse. Sunburn (erythema or redness)in a subregion qualified for inclusion in the ‘with sunburn’ column inthe table.

The data in the above Table 5 indicate that β-damascenone providessignificant sunburn protection when given by mouth as 4 equal doses of20 uL and as a single dose of 20 uL. Four equal doses of 10 uL was notsignificant but was borderline, indicating that a significant resultmight occur with more mice. Groups 2 and 4 were replications and showedclose agreement and their combined comparison to UVR-only is highlysignificant. These results establish clearly that sunburn is preventedefficaciously at 5 days after either a single dose of 20 uL or 4 dailydoses of 20 uL of oral β-damascenone. Other data not shown indicatesunburn prevention persists for at least 14 days after the 4 day regimenof terpene whether oral or topical administration.

The following endpoints have been (1 and 4) or will be (2 and 3)measured at the times indicated in Table 4:

1. Skin and epidermal thickness (as measured by histology);

2. Basal cell proliferation rate (as measured by a PCNA technique);

3. Epidermal cell gene expression (as measured by a microarrayanalysis); and

4. Sunburn protection (as measured by skin redness in response tostandard UVR exposure).

Example 5

Effect of d-limonene healing of atopic dermatitis when presented as drycracked skin

Subjects

Four healthy participating female volunteers ages 40-70 years who wereall experiencing dry cracked skin on hands and feet especially in wintermonths which in some instances were bleeding were enrolled. Their skinwould be one of the four skin types I, II, III and IV.

Compound and Dose

d-limonene was ingested either in the form of a gel capsule or added tojuice as liquid. Two (2) subjects took 2 g gel capsule every 24 hoursfor 4 days and two (2) subjects took 0.2 g every 24 hours for 4 days asliquid in their orange juice.

Results

The dry cracked skin on the finger tips and heels started to heel 4-5days after the initial dose was given. The skin became smoother withouta rough surface. Cracks were eliminated completely. The d-limoneneeffect lasted for about 14 to 18 days after which the symptoms returned.Similar results were obtained when the volunteers repeated the intakewith a new dose of d-limonene. This observations were made by theparticipants themselves and they were not evaluated by a physician.

Number of participants: 4 Age of participants: 40-70 Gender: Females 4Males 0 Duration of experiment: 14-18 days Dose: 0.2 or 2 g ofd-limonene Evaluation: Self-examination of the fingers and feetperformed by participants. Effectiveness: 100%

Example 7

TABLE 6 Comparing the thickness of nucleated keratinocyte portion of theepidermis versus the stratum corneum of Skh1 mouse skin. Thicknessmeasurements were based on computer projections of photomicrographs ofregular H&E histological sections taken at various times after regimensof β-damascenone administered orally by feeding tube. Living Epidermis(arbitrary units) Stratum Corneum (arbitrary units) Group RegimenThickness StDev p-value Thickness StDev p-value 1 Control 32.2 5.2 — —9.00 3.5 — — 2 20 μL, ×1, 1 d, 20 35.0 5.0 0.40 NS 20.0 15.0 0.11 NS 310 μL, ×4, 1 d, 40 37.0 2.7 0.12 NS 15.3 10.1 0.19 NS 4 20 μL, ×4, 1 d,80 36.0 5.6 0.38 NS 17.8 9.2 0.062 BLS 5 80 μL, ×1, 1 d, 80 36.0 5.60.28 NS 16.3 5.7 0.044 S 6 20 μL, ×1, 5 d, 20 38.5 10.3 0.21 NS 37.0 8.10.0001 HS 7 10 μL, ×4, 5 d, 40 40.2 13.2 0.19 NS 20.2 6.7 0.008 HS 8 80μL, ×1, 5 d, 80 36.2 4.3 0.18 NS 25.5 6.7 0.0008 HS 9 20 μL, ×4, 5 d, 8032.5 6.5 0.92 NS 21.2 6.3 0.004 HS 10 10 μL, ×4, 12 d, 40 30.7 10.1 0.75NS 18.7 12.1 0.095 NS 11 20 μL, ×4, 12 d, 80 23.3 2.9 0.015 S* 21.7 2.90.001 HS Average (Groups 2-11) 34.6 7.4 — — 21.0 4.6 — — HS = highlySignificant; S = Significant; NS = Not Significant; BLS = Border-lineSignificant; S* = Significant (but a reduction and not relevant). Note1: p-values are based on Student's t-distribution, degrees of freedomrange: 7 to 8. Note 2: The 3 items separated by commas in Regimen are 1)administered quantity (μL), 2) number of daily applications, 3) time ofhistological section after the final β-damascenone administration (d)and 4) total dose in μL.

The data in Table 6 indicate highly significant increases in thethickness of the stratum corneum at day 5 after the β-damascenoneexposures were completed but no corresponding increase was observedamong the nucleated keratinocytes in the epidermis, i.e. the epidermalthickness is substantially increased by the β-damascenone, but theentire increase can be ascribed solely to the increased thickness of thestratum corneum. Generally as the β-damascenone dose increased, theincreased thickness of the stratum corneum occurred earlier andpersisted longer. For example, at a single dose of 20 μL, no increasedthickness was noted a day 1, but significantly increased thickness wasoccurred at day 5. At the next higher dose of 40 μL (10 μL×4), a similarpattern was seen, i.e. no thickness increase (borderline p-value) at day1 and a highly significant increase at day 5, but followed by a returnto no significant increase at day 12 indicating a response reversalbetween day 5 and day 12. At the highest dose of 80 μL (20 μL×4) thepattern of no significant increase at day 1 and a highly significantincrease at day 5 persisted, but now a highly significant increaseoccurred at 12 days also, i.e. the reversal at the lower dose wasovercome at the higher dose. A single dose of 80 μL was by far the mostpotent in that a significant increase of stratum thickness wasdocumented even at day 1. None of the exposure regimens, including thesingle dose of 80 μL, produced any detectible increase in the nucleatedkeratinocytes of the epidermis at any dose or time point.

The β-damascenone acted specifically to increase the thickness of thestratum corneum without increasing the population of nucleatedkeratinocytes. Studies are in progress to evaluate the possibility thata proliferative stimulation of the keratinocytes may have occurred butwas too small to be observed as a significant increase in totalnucleated cell number. The method will utilize an antibody toproliferating cell nuclear antigen (PCNA) as a measure of the rate ofkeratinocyte proliferation on histological sections of the same tissuesutilized for evaluating the histological responses. Also gene expressionexperiments are in progress to evaluate how the induction oflate-cornifying genes, such as filaggrin, loricrin, keratin, etc. dependon the amount and frequency of β-damascenone treatments whether byingestion or by topical application.

Similar Studies Will be Extended to Other Terpenes.

The data presented here indicate a very unusual and heretofore unknownproperty of terpenes, such as β-damascenone, of being able to alter thestructure of the stratum corneum without a concomitant increase in thenumber of nucleated keratinocytes. There are substantial practicalbenefits that might become available, because these compounds aregenerally regarded as safe and can be given at higher doses ifindicated, whereas currently available treatments for conditions likeatopic dermatitis rely heavily on retinoid-type compounds that are knownto be anti-inflammatory. The latter compounds cannot be administered atefficacious doses because of their inherent toxicity.

It will be apparent to those of skill in this art that the foregoingexamples are only illustrative of the present invention and thatmodifications and additions are apparent to those of skill in the art.All such modifications and additions are included within the scope ofthe present invention, which is to be limited only by the scope,including equivalents, of the following, appended claims.

We claim:
 1. A method of restoring, generating or regenerating a stratumcorneum or stratum granulosum comprising administering a terpene or aterpenoid selected from the group consisting of a monoterpene, asesquiterpene, a norisoprenoid, beta-damascenone, nootkatone, andd-limonene in a dosage sufficient to increase the thickness of thestratum corneum, increase skin thickness, decrease or eliminate thenumber or size of breaches in the stratum corneum, or decrease theamount of moisture or fluid that may pass through the skin in a givenperiod of time, and wherein the terpene or terpenoid is administered ina dosage and in amounts sufficient to increase epidermal cellproliferation and increase keratin production in the skin of subjectsreceiving the terpene or terpenoid compared to the amount of epidermalcell proliferation and keratin production present in the skin ofsubjects not receiving the terpene or terpenoid.
 2. A method accordingto claim 1 wherein the terpene or terpenoid is administered at least onetime per day.
 3. A method according to claim 1 wherein the terpene orterpenoid is administered orally.
 4. A method according to claim 1wherein the terpene or terpenoid is administered topically.
 5. A methodaccording to claim 1 wherein the terpene or terpenoid is administered ina dosage and in amounts sufficient to alter the expression of one ormore gene products selected from the group consisting of NFKappaB, COX1,COX2, MAPKS, PGE2, ODC, protein kinase C, INOS, PI3-AKT, TNF-A, AP1,NRF2, KEAP1 and IKappa kinase.